Immunological control of beta-amyloid levels in vivo

ABSTRACT

The present invention provides an antibody which catalyzes hydrolysis of β-amyloid at a predetermined amide linkage. The antibody preferentially binds a transition state analog which mimics the transition state adopted by β-amyloid during hydrolysis at a predetermined amide linkage and also binds to natural β-amyloid with sufficient affinity to detect by ELISA. Alternatively, the antibody preferentially binds a transition state analog which mimics the transition state adopted by β-amyloid during hydrolysis at a predetermined amide linkage, and does not bind natural β-amyloid with sufficient affinity to detect by ELISA. Antibodies generated are characterized by the amide linkage which they hydrolyze. Specific antibodies provided include those which catalyze the hydrolysis at the amyloid linkages between residues 39 and 40, 40 and 41, and 41 and 42, of β-amyloid. The present invention also provides a vectorized antibody which is characterized by the ability to cross the blood brain barrier and is also characterized by the ability to catalyze the hydrolysis of β-amyloid at a predetermined amide linkage. The vectorized antibody can take the form of a bispecific antibody, which has a first specificity for the transferrin receptor and a second specificity for a transition state adopted by β-amyloid during hydrolysis. The present invention also provides a method for sequestering free β-amyloid in the bloodstream of an animal by intravenously administering antibodies specific for β-amyloid to the animal in an amount sufficient to increase retention of β-amyloid in the circulation. In addition, the present invention provides a method for sequestering free β-amyloid in the bloodstream of an animal by immunizing an animal with an antigen comprised of an epitope which is present on β-amyloid endogenous to the animal under conditions appropriate for the generation of antibodies which bind endogenous β-amyloid. Therapeutic applications of these methods include treating patients diagnosed with, or at risk for Alzheimer&#39;s disease. Methods for reducing levels of β-amyloid in the brain of an animal, by intravenously administering antibodies specific for endogenous β-amyloid to the animal, or by immunizing the animal with an antigen comprised of an epitope which is present on endogenous β-amyloid are also provided. In one embodiment, the antigen used to generate the antibodies is a transition state analog which mimics the transition state adopted by β-amyloid during hydrolysis at a predetermined amide linkage. Similar methods which utilize or generate antibodies which catalyze the hydrolysis of β-amyloid for reducing levels of circulating β-amyloid in an animal, and also for preventing the formation of amyloid plaques in the brain of an animal, and also for disaggregating amyloid plaques present in the brain of an animal, are also provided. Also provided is a method for generating antibodies which catalyze hydrolysis of a protein or polypeptide by immunizing an animal with an antigen comprised of an epitope which has a statine analog which mimics the conformation of a predetermined hydrolysis transition state of the polypeptide. A similar method, which utilizes reduced peptide bond analogs to mimic the conformation of a hydrolysis transition state of a polypeptide, is also provided.

BACKGROUND OF THE INVENTION

[0001] Alzheimer's disease is a progressive and ultimately fatal form ofdementia that affects a substantial portion of the elderly population.Definitive diagnosis at autopsy relies on the presence ofneuropathological brain lesions marked by a high density of senileplaques. These extracellular deposits are found in the neo-cortex,hippocampus and amygdala as well as in the walls of the meningeal andcerebral blood vessels. The principal component of these plaques is a 39to 43 residue β-amyloid peptide. Each plaque contains approximately 20fmole (80 picograms) of this 4 kDa peptide (Selkoe et al., J. ofNeurochemistry 46: 1820 (1986)). Apolipoprotein E and neurofibrillarytangles formed by the microtubule-associated tau protein are also oftenassociated with Alzheimer's disease.

[0002] β-amyloid is proteolytically cleaved from an integral membraneprotein called the β-amyloid precursor protein. The gene which codes forthis protein in humans is found on chromosome 21 (St George-Hyslop etal., Science 235: 885 (1987), Kang et al., Nature 325: 733 (1987)).Numerous cultured cells and tissues (eg. brain, heart, spleen, kidneyand muscle) express this β-amyloid precursor protein and also secretethe 4 kDa β-amyloid fragment into culture media, apparently as part of anormal processing pathway.

[0003] While it is difficult to establish an absolute causalrelationship between β-amyloid or the plaques it forms and Alzheimer'sdisease, there is ample evidence to support the pathogenic role ofβ-amyloid. For example, patients with Down's syndrome have an extra copyof the β-amyloid precursor protein gene due to trisomy of chromosome 21(St George-Hyslop et al., Science 235: 885 (1987), Kang et al., Nature325: 733 (1987)). They correspondingly develop an early-onsetAlzheimer's disease neuropathology at 30-40 years of age. Moreover,early-onset familial Alzheimer's disease can result from mutations inthe β-amyloid precursor protein gene which fall within or adjacent tothe β-amyloid sequence (Hardy, J., Nature Genetics 1: 233 (1992)). Theseobservations are consistent with the notion that deposition of β-amyloidas plaques in the brain are accelerated by an elevation in itsextracellular concentration (Scheuner et al., Nature Med. 2: 864(1996)). The finding that β-amyloid is directly neurotoxic both in vitroand in vivo (Kowall et al., Proc. Natl. Acad. Sci. 88: 7247 (1991)),suggest that soluble aggregated β-amyloid, not the plaques per se, mayproduce the pathology.

[0004] Observations have indicated that amyloid plaque formation mayproceed by a crystallization type mechanism (Jarrett et al., Cell 73:1055 (1993)). According to this model, the seed that initiates plaquenucleation is an β-amyloid which is 42 or 43 amino acids long (Aβ₁₋₄₃).The rate-determining nucleus formed by Aβ₁₋₄₃ or Aβ₁₋₄₂ allows peptidesAβ₁₋₄₀ or shorter to contribute to the rapid growth of an amyloiddeposit. This nucleation phenomenon was demonstrated in vitro by theability of Aβ₁₋₄₂ to cause the instantaneous aggregation of akinetically stable, supersaturated solution of Aβ₁₋₄₀. That finding hasled to the possibility that Aβ₁₋₄₀ might be relatively harmless in theabsence of the nucleation peptides Aβ₁₋₄₂ or Aβ₁₋₄₃. Indeed, elevatedlevels of these long peptides have been found in the blood of patientswith familial Alzheimer's disease (Scheuner et al., Nature Med. 2: 864(1996)). Moreover, Aβ₁₋₄₂ or Aβ₁₋₄₃ was found to be the predominant formdeposited in the brain plaques of many Alzheimer's disease patients(Gravina et al., J. of Biol. Chem. 270: 7013 (1995)).

[0005] Given the central role played by β-amyloid, it has becomeincreasingly important to understand the interrelationship between thedifferent pools of these molecules in the body. Free β-amyloid presentin the blood most likely arises from peptide released by proteolyticcleavage of β-amyloid precursor protein present on cells in theperipheral tissues. Likewise most of the free β-amyloid found in thebrain and cerebrospinal fluid is probably derived from peptide releasedby secretase cleavage of β-amyloid precursor protein expressed on braincells. The peptides are identical regardless of origin, and the resultsfrom several studies suggest an intercommunication between these pools.

SUMMARY OF THE INVENTION

[0006] One aspect of the present invention is an antibody whichcatalyzes hydrolysis of β-amyloid at a predetermined amide linkage. Inone embodiment, the antibody preferentially binds a transition stateanalog which mimics the transition state adopted by β-amyloid duringhydrolysis at a predetermined amide linkage and also binds to naturalβ-amyloid with sufficient affinity to detect using an ELISA. In anotherembodiment, the antibody preferentially binds a transition state analogwhich mimics the transition state adopted by β-amyloid during hydrolysisat a predetermined amide linkage, and does not bind natural β-amyloidwith sufficient affinity to detect using an ELISA. Antibodies generatedare characterized by the amide linkage which they hydrolyze. Specificantibodies include those which catalyze the hydrolysis at the amyloidlinkages between residues 39 and 40, 40 and 41, and 41 and 42, ofβ-amyloid.

[0007] Another aspect of the present invention is a vectorized antibodywhich is characterized by the ability to cross the blood brain barrierand is also characterized by the ability to catalyze the hydrolysis ofβ-amyloid at a predetermined amide linkage. In one embodiment, thevectorized antibody is a bispecific antibody. Preferably, the vectorizedantibody has a first specificity for the transferrin receptor and asecond specificity for a transition state adopted by β-amyloid duringhydrolysis. Specific vectorized antibodies include those which catalyzethe hydrolysis at the amyloid linkages between residues 39 and 40, 40and 41, and 41 and 42, of β-amyloid.

[0008] Another aspect of the present invention is a method forsequestering free β-amyloid in the bloodstream of an animal byintravenously administering antibodies specific for β-amyloid to theanimal in an amount sufficient to increase retention of β-amyloid in thecirculation. Therapeutic applications of this method include treatingpatients diagnosed with, or at risk for Alzheimer's disease.

[0009] Another aspect of the present invention is a method forsequestering free β-amyloid in the bloodstream of an animal byimmunizing an animal with an antigen comprised of an epitope which ispresent on β-amyloid endogenous to the animal under conditionsappropriate for the generation of antibodies which bind endogenousβ-amyloid. Therapeutic applications of this method include treatingpatients diagnosed with, or at risk for Alzheimer's disease.

[0010] Another aspect of the present invention is a method for reducinglevels of β-amyloid in the brain of an animal by intravenouslyadministering antibodies specific for endogenous β-amyloid to the animalin an amount sufficient to increase retention of β-amyloid in thecirculation of the animal. In one embodiment, the antibodies arecatalytic antibodies which catalyze hydrolysis of β-amyloid at apredetermined amide linkage. The antibodies may be either monoclonal orpolyclonal. In one embodiment, the antibodies specifically recognizeepitopes on the C-terminus of β-amyloid₁₋₄₃.

[0011] Another aspect of the present invention is a method for reducinglevels of β-amyloid in the brain of an animal, by immunizing the animalwith an antigen comprised of an epitope which is present on endogenousβ-amyloid under conditions appropriate for the generation of antibodieswhich bind endogenous β-amyloid. In one embodiment, the antigen is atransition state analog which mimics the transition state adopted byβ-amyloid during hydrolysis at a predetermined amide linkage. In apreferred embodiment, the antigen is comprised of Aβ₁₀₋₂₅. Preferably,the antibodies generated have a higher affinity for the transition stateanalog than for natural β-amyloid, and catalyze hydrolysis of endogenousβ-amyloid.

[0012] Similar methods which utilize or generate antibodies whichcatalyze the hydrolysis of β-amyloid for reducing levels of circulatingβ-amyloid in an animal, and also for preventing the formation of amyloidplaques in the brain of an animal, are also provided. Also, methods fordisaggregating amyloid plaques present in the brain of an animal byutilizing or generating antibodies which catalyze the hydrolysis ofβ-amyloid are provided.

[0013] Another aspect of the present invention is a method fordisaggregating amyloid plaques present in the brain of an animal byintravenously administering vectorized bispecific antibodies to theanimal in an amount sufficient to cause significant reduction inβ-amyloid levels in the brain of the animal. The vectorized bispecificantibodies are competent to transcytose across the blood brain barrier,and have the ability to catalyze hydrolysis of endogenous β-amyloid at apredetermined amide linkage upon binding. Preferably, the vectorizedbispecific antibodies specifically bind the transferrin receptor.

[0014] Another aspect of the present invention is a method forgenerating antibodies which catalyze hydrolysis of a protein orpolypeptide by immunizing an animal with an antigen comprised of anepitope which has a statine analog which mimics the conformation of apredetermined hydrolysis transition state of the polypeptide, underconditions appropriate for the generation of antibodies to thehydrolysis transition state. This method can be used to generatecatalytic antibodies to β-amyloid. A similar method, which utilizesreduced peptide bond analogs to mimic the conformation of a hydrolysistransition state of a polypeptide, is also provided.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1 is an amino acid sequence listing (SEQ ID NO: 1) of the 43residue β-amyloid peptide (Aβ).

[0016]FIG. 2 is an amino acid sequence listing (SEQ ID NO: 2) of theantigenic peptide made from the N-terminal sequence of β-amyloid(Aβ₁₋₁₆).

[0017]FIG. 3 is an amino acid sequence listing (SEQ ID NO: 3) of theantigenic peptide made from the central region of β-amyloid (Aβ₁₀₋₂₅).

[0018]FIG. 4 is an amino acid sequence listing (SEQ ID NO: 4) (Aβ₃₅₋₄₃)of the antigenic peptide made from the C-terminal sequence of β-amyloid.

[0019]FIG. 5 is a diagrammatic representation of data from an ELISAcomparing monoclonal antibody binding to Aβ₃₅₋₄₃ and Aβ₁₋₄₃ versusAβ₁₋₄₀.

[0020]FIG. 6 indicates the amide linkages in the peptide made from theβ-amyloid C-terminal sequence (SEQ ID NO: 4) that were independentlyreplaced with a statyl moiety, to generate the different statinetransition state analogs of the peptide.

[0021]FIG. 7 indicates the amide linkages in the peptide made from theβ-amyloid central sequence (SEQ ID NO: 3) that were independentlyreplaced with a statyl moiety, to generate the different phenylalaninestatine transition state analogs of the peptide.

[0022]FIG. 8 is a structural comparison between the native β-amyloidpeptide and the transition state phenylalanine statine β-amyloid peptideanalog.

[0023]FIG. 9 is a structural comparison between the native β-amyloidpeptide and the reduced peptide bond transition state β-amyloid peptideanalog.

[0024]FIG. 10 is a formulaic representation of the native C-terminalregion of β-amyloid, and the phosphonamidate transition state analog ofthe C-terminal region of β-amyloid (Aβ₃₅₋₄₃).

[0025]FIG. 11 indicates the putative transition state for peptidehydrolysis by zinc peptidases, compared to the phosphonate andphosphonamidate mimics.

[0026]FIG. 12 is a structural comparison of the native β-amyloid peptideand the transition state phosphonamidate β-amyloid peptide which has thepeptide link between Gly 38 and Val 39 replaced with a phosphonamidatebond.

[0027]FIG. 13 is a diagrammatic representation of data from an ELISAwhich assess the binding of monoclonal antibodies generated totransition state β-amyloid peptide analogs, to the normal Aβ₁₋₄₃ and tothe phenylalanine statine transition state β-amyloid peptide.

[0028]FIG. 14 is a diagrammatic representation of data from an ELISAcomparing antibody binding to the statine transition state β-amyloidpeptide versus native Aβ₁₋₄₃ and native Aβ₁₋₄₀.

[0029]FIG. 15 is a graph of data showing the cleavage of¹²⁵I-Aβ-sepharose by monoclonal antibodies generated to transition stateanalogs of β-amyloid.

[0030]FIG. 16 is a diagrammatic representation of data which quantitatethe attachment of bispecific antibody to receptor-positive cells.

[0031]FIG. 17 is a diagrammatic representation of data obtained fromexperiments designed to track the transcytosis of vectorized bispecificantibody into brain.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to immunologically based methodsfor controlling levels of β-amyloid in the body of an animal. Theinvention is based on the finding that antibodies specific for β-amyloidare able to bind β-amyloid in the presence of a physiological level ofhuman serum albumin. The invention is also based on the finding that ananimal can tolerate the presence of antibodies specific for β-amyloid inamounts sufficient to sequester β-amyloid in the bloodstream.

[0033] One aspect of the present invention relates to a method forsequestering free β-amyloid in the bloodstream of an animal. The solubleand insoluble forms of β-amyloid present within an animal are in dynamicequilibrium. Soluble β-amyloid is thought to translocate between bloodand cerebrospinal fluid. Insoluble β-amyloid aggregates deposit from thesoluble pool in the brain, as amyloid plaques. Results detailed in theExemplification section below indicate that intravenous administrationof antibodies specific for β-amyloid to an animal impedes the passage ofsoluble β-amyloid out of the peripheral circulation. This occurs becausethe β-amyloid specific antibodies, which are restricted to theperipheral circulation, bind to β-amyloid and sequester it in thecirculation. Such sequestration is accomplished through intravenousadministration of an appropriate amount of antibodies specific forβ-amyloid to the animal. The amount of antibody which is sufficient toproduce sequestration is dependent upon various factors (e.g., specificcharacteristics of the antibody to be delivered, the size, metabolism,and overall health of the animal) and are to be determined on a case bycase basis.

[0034] Administered antibodies can be monoclonal antibodies, a mixtureof different monoclonal antibodies, polyclonal antibodies, or anycombination therein. In one embodiment, the antibodies bind to theC-terminal region of β-amyloid. Such antibodies specifically bind theless abundant, but more noxious Aβ₁₋₄₃ species in the blood as opposedto the smaller and less detrimental Aβ₁₋₄₀. In another embodiment, acombination of antibodies having specificity for various regions ofβ-amyloid are administered. In another embodiment, antibodies whichcatalyze the hydrolysis of β-amyloid, discussed in more detail below,are administered either alone or in combination with otheranti-β-amyloid antibodies.

[0035] The animal to which the antibodies are administered is any animalwhich has circulating soluble β-amyloid. In one embodiment, the animalis a human. The human may be a healthy individual, or alternatively, maybe suffering from or at risk for a disease in which elevated β-amyloidlevels are thought to play a role, for example a neurodegenerativedisease such as Alzheimer's disease.

[0036] A related aspect of the present invention is a method forsequestering free β-amyloid in the bloodstream of an animal bystimulating an immune response within the animal to endogenousβ-amyloid. The results detailed in the Exemplification below indicatethat an animal can tolerate the induction of an immune response whichproduces antibodies to endogenous β-amyloid, and that the presence ofsuch antibodies will alter the distribution of β-amyloid in the body, ina similar manner as the above described method of administeringβ-amyloid binding antibodies. The immune response to endogenousβ-amyloid is generated by immunizing the animal with one or moreantigens comprised of epitopes present on the endogenous β-amyloid.Epitopes present on the inoculated antigens can correspond to epitopespresent on any region of the β-amyloid molecule. In a preferredembodiment, epitopes found on the C-terminal region of β-amyloid areused to generate antibodies which specifically bind the Aβ₁₋₄₃ speciesas opposed to the smaller Aβ₁₋₄₀. In an alternate embodiment, acombination of different epitopes are administered to generate a varietyof antibodies to β-amyloid. A more generalized immune response isgenerated by immunizing either with a mixture of different small peptideantigens or with the full-length 43 residue β-amyloid peptide. Inanother embodiment, antigens used for inoculation include transitionstate analogs of β-amyloid peptides to induce antibodies which havecatalytic activity directed towards β-amyloid hydrolysis, described indetail below.

[0037] The immunoreactivity of the antigens can be enhanced by a varietyof methods, many of which involve coupling the antigen to an immunogeniccarrier. In addition, various methods are known and available to one ofskill in the art for specifically enhancing the immunogenicity ofendogenous molecules or the epitopes contained therein. Variousmodifications can be made to the β-amyloid antigen(s) described hereinto render it more compatible for human use. For example, the peptide(s),can be genetically engineered into appropriate antigenic carriers, orDNA vaccines can be designed.

[0038] The above techniques for sequestering β-amyloid in thecirculation are also useful for reducing the levels of β-amyloid in thebrain. Because the formation of amyloid plaques in the brain isdependent, at least in part, on the levels of free β-amyloid present inthe brain, reducing brain β-amyloid levels of an animal will, in turn,reduce the formation of amyloid plaques in the brain. Therefore, theabove techniques are useful for preventing the formation of amyloidplaques in the brain of an animal. This is especially applicable to ananimal which is considered at risk for the development of amyloidplaques; a risk which may result from a genetic predisposition or fromenvironmental factors. Administration of antibodies, or immunization ofthe animal to produce endogenous antibodies, to β-amyloid can be oftherapeutic benefit to such an animal (e.g., a human who has a familyhistory of Alzheimer's disease, or who is diagnosed with the disease).

[0039] Another aspect of the present invention relates to antibodieswhich are characterized by the ability to catalyze the hydrolysis ofβ-amyloid at a predetermined amide linkage. Experiments detailed in theExemplification section demonstrate the generation of differentantibodies which have proteolytic activity towards β-amyloid. Suchantibodies are generated by immunizing an animal with an antigen whichis a transition state analog of the β-amyloid peptide. A transitionstate analog mimics the transition state that β-amyloid adopts duringhydrolysis of a predetermined amide linkage. Transition state analogsuseful for generating the catalytic antibodies include, withoutlimitation, statine, phenylalanine statine, phosphonate,phosphonamidate, and reduced peptide bond transition state analogs.

[0040] Antibodies generated to epitopes unique to the transition statepreferentially bind β-amyloid in the transition state. Binding of theseantibodies stabilizes the transition state, which leads to hydrolysis ofthe corresponding amide bond. The particular amide linkage to behydrolyzed is chosen based upon the desired cleavage product. Forexample, cleavage of full length β-amyloid into two peptide fragmentswhich cannot aggregate into amyloid plaques would be of therapeutic usein the methods disclosed herein. Antibodies may be either monoclonal orpolyclonal. Several such transition state mimics have been made and usedas antigen in the generation of monoclonal antibodies which catalyze thecleavage at the indicated linkage. These antigens and the antibodiesgenerated are listed in Table 8 of the Exemplification section below.Antibodies generated to antigens which have transition state mimicsincorporated at a specific amide linkage, should bind the naturalhydrolysis transition states of these linkages in native β-amyloid,stabilizing the transition state and catalyzing cleavage at thatlinkage.

[0041] At least two different classes of antibodies are generated by theabove methods. The first class preferentially binds the transition stateanalog, and also detectably cross reacts with natural β-amyloid usingthe ELISA detailed in the Exemplification section, to detect binding.The second class binds the transition state analog, and does notdetectably cross react with natural β-amyloid using the ELISA proceduredetailed in the Exemplification section to detect binding. Both classesof antibodies have potential value as catalytic antibodies. Therespective binding affinities of an anti-transition state antibody islikely to reflect its activity at catalyzing hydrolysis. It is thoughtthat in order for an antibody to have activity at catalyzing hydrolysisof a protein, it must possess at least a minimal ability to bind thenatural (non-transition) state of the protein. Antibodies which retainsignificant binding for β-amyloid, (that strongly cross react withnatural β-amyloid) may be more efficient at catalyzing hydrolysis due toa higher efficiency of binding the β-amyloid. Once bound, theseantibodies force the protein into a transition state conformation forhydrolytic cleavage. Alternatively, antibodies which only minimallycross react with natural β-amyloid, although less efficient at bindingnative β-amyloid, are likely to be more efficient at forcing the boundβ-amyloid into the transition state conformation for hydrolyticcleavage. It should be pointed out that failure to detect binding of theanti-transition state antibodies to natural β-amyloid by the ELISAmethods presented in the Exemplification herein does not necessarilyreflect an inability to bind natural β-amyloid sufficiently to functionas a catalytic antibody. More likely, a lack of detection merelyreflects the sensitivity limitations of the assay.

[0042] Antibodies which have substantial affinity for the predictedcleavage products of the native β-amyloid peptide may be subject toproduct inhibition and might therefore exhibit low turnover. Suchundesirable antibodies can be identified by secondary screening usingpeptides which contain epitopes of the predicted cleavage products(e.g., via ELISA).

[0043] In a preferred embodiment, the antibodies are monoclonal.Monoclonal antibodies are produced by immunizing an animal (e.g., mouse,guinea pig, or rat) with the transition state analog antigen, andsubsequently producing hybridomas from the animal, by standardprocedures. Hybridomas which produce the desired monoclonal antibodiesare identified by screening. One example of a screening method ispresented in the Exemplification section which follows. In anotherembodiment, the antibodies are polyclonal. Polyclonal antibodies aregenerated by immunizing an animal (e.g., a rabbit, chicken, or goat)with antigen and obtaining sera from the animal. Polyclonal antibodieswhich have the desired binding specificities can be further purifiedfrom the sera by one of skill in the art through the course of routineexperimentation.

[0044] Catalytic antibodies specific for β-amyloid can alternatively begenerated in an individual through the use of anti-idiotype vaccinesdesigned to elicit the production of catalytic antibodies. Such vaccinesare described in the disclosure of Raso and Paulus (U.S. patentapplication Ser. No. 09/102,451, ANTI-IDIOTYPE VACCINES TO ELICITCATALYTIC ANTIBODIES, filed by Applicants Jun. 22, 1998, currentlypending), the contents of which are incorporated herein by reference.

[0045] Another aspect of the present invention is the use of statine andreduced peptide bond analogs to elicit catalytic antibodies havingproteolytic activity. The Exemplification section below details methodsfor using statine analogs as antigen in the production of catalyticantibodies, and also lists examples of anti-transition-state antibodiesgenerated using these methods. The “statyl” moiety is derived fromnaturally evolved protease transition state inhibitors like amastatin,pepstatin, and bestatin. These naturally-occurring statine-basedinhibitors have been used to effectively block the activity ofaminopeptidases, aspartic proteases and the HIV protease. Syntheticpeptides containing a statine residue offer novel features for theinduction of catalytic antibodies. The statyl moiety has a tetrahedralbond geometry, its length is extended by two CH₂ units, it has astrategically placed OH group and the structure has no charge. Thepresence of the additional CH₂ units is expected to elicit a moreelongated antibody combining site, and antibodies possessing thisextended site will induce extra strain on the peptide substrate,producing an accelerated catalysis. In addition, the —OH group in thesestatine analogs is thought to better approximate the position andchemistry of the true transition state. Statine-based transition-stateanalogs should therefore elicit a class of antibodies which issignificantly different from those obtained from the more commonly usednegatively charged phosphonate analogs.

[0046] Reduced peptide bond analogs introduce a tetrahedralconfiguration, without increasing the distance between amino acidresidues. This feature should more closely approximate the truetransition state geometry, than previously used analogs. A positivelycharged secondary amine replaces the amide nitrogen of the naturalpolypeptide and should elicit a complementary negatively charged sidechain at a proximal locus in the antibody combining site. The presenceof such ancillary glutamyl or aspartyl groups on the antibody willassist antibody-mediated catalysis of peptide cleavage via acid-baseexchange. Reduced peptide bond-based transition-state analogs shouldtherefore elicit a class of antibodies which is significantly differentfrom those obtained from using the more commonly used negatively chargedphosphonate analogs. Reduced peptide bond analogs and statine analogscan be used to produce specific transition state analog antigens for awide variety of proteins or polypeptides. These antigens can in turn beused to generate the respective catalytic antibodies.

[0047] Administration of the β-amyloid catalytic antibodies describedabove can be used in the methods described above for 1) sequesteringfree β-amyloid in the bloodstream of an animal, 2) reducing levels ofβ-amyloid in the brain of an animal, and 3) preventing the formation ofamyloid plaques in the brain of an animal, to generate the analogousresults. Experiments presented in the Exemplification demonstrate thatimmunization of an animal with a transition state analog results in thegeneration of an immune response to produce antibodies which recognizethe transition state, and which catalyze hydrolysis of the β-amyloidprotein. This indicates that the transition state analogs can be used asantigens in these methods to induce the production of antibodies in theanimal which recognize and catalyze cleavage of endogenous β-amyloid.

[0048] Methods which involve reducing overall levels of β-amyloid in ananimal through the proteolytic action of the above described catalyticantibodies are also encompassed by the present invention. The presenceof functional catalytic antibodies in the circulation of an animal willreduce the level of intact β-amyloid in the circulation by selectivehydrolytic cleavage. Accordingly, the present invention provides amethod for reducing levels of circulating β-amyloid in an animal byintroducing the above described catalytic antibodies into the animal.Administration of the antibodies to the animal is preferably viaintravenous administration. Such antibodies are either monoclonal, mixedmonoclonal, polyclonal or any mixture thereof. The origin of theantibody may affect the half-life of the antibody in the animal;antibodies from less related species are more likely to be recognized asforeign by the animal's immune system. Preferably, administeredantibodies are derived from a species closely related to the animal, tomaximize half-life and minimize adverse reactions by the host.Administration of isolated variable region antibody fragments mayproduce beneficial results in this regard.

[0049] The present invention also provides a method for reducing levelsof circulating β-amyloid in an animal by immunizing the animal with aβ-amyloid transition state analog to induce endogenous catalyticantibody production. The use and design of such vaccines is describedabove, and detailed in the Exemplification section below.

[0050] The reduction of β-amyloid levels in the circulation of an animalis expected to displace the equilibrium of β-amyloid in the body, andultimately lead to a reduction in the levels of β-amyloid in the brainof the animal through mass action. In this respect, the presentinvention provides methods for reducing the levels of β-amyloid in thebrain of an animal, by either administering catalytic antibodies to theanimal, or by administering a transition state analog to induceendogenous antibody production. It follows that these procedures alsohave value as methods for preventing the formation of amyloid plaques inthe brain of an animal, since the resulting reduction in the levels ofβ-amyloid in the brain of an animal should prevent the formation ofamyloid plaques. These procedures also have value as methods fordisaggregating amyloid plaques present in the brain of an animal, sinceevidence indicates that lower brain β-amyloid levels can lead to thedisaggregation of plaques.

[0051] Another aspect of the present invention provides a more directmethod of altering the distribution of β-amyloid in the brain byactually delivering anti-β-amyloid antibodies to the brain. Methodsdescribed above for reducing levels of β-amyloid in the brain and forpreventing aggregation of amyloid plaques depend upon exchange betweenβ-amyloid pools in the blood, tissues, cerebrospinal fluid and thebrain, the exchange being driven by an antibody-mediated disruption ofthe equilibrium between these different pools. In contrast, delivery ofanti-β-amyloid antibodies to the brain will directly affect β-amyloidaggregation. Evidence presented in the Exemplification section belowindicates that the binding of certain anti-β-amyloid antibodies inhibitsthe initial aggregation of β-amyloid in vitro, and also disaggregatespreformed in vitro β-amyloid complexes. Moreover, if insoluble peptideis in equilibrium with a low level of soluble β-amyloid, then ananti-β-amyloid binding antibody could upset this balance and graduallydissolve the precipitate. These observations indicate that the presenceof β-amyloid antibodies in the brain will directly inhibit the formationof amyloid plaques and will also disaggregate preformed plaques bydisrupting the dynamic equilibrium between soluble β-amyloid andfibrillar β-amyloid deposited as plaques. Furthermore, a highly activecatalytic antibody is expected to destroy insoluble β-amyloid plaques byhydrolytically cleaving the constituent aggregated peptides.

[0052] One way of delivering antibodies to the brain is by producingvectorized antibodies competent for transcytosis across the blood-brainbarrier. Vectorized antibodies are produced by covalently linking anantibody to an agent which promotes delivery from the circulation to apredetermined destination in the body. Examples of vectorized moleculeswhich can traverse the blood-brain barrier are found in the prior art(Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-2622 (1993);Broadwell et al., Exp. Neurol. 142: 47-65 (1996)). In these examples,antibodies are linked to another macromolecule, the antibodies being theagent which promotes delivery of the macromolecules. One example of suchan agent is an antibody which is directed towards a cell surfacecomponent, such as a receptor, which is transported away from the cellsurface. Examples of antibodies which confer the ability to trancytosethe blood-brain barrier include, without limitation, anti-insulinreceptor antibodies, and also anti-transferrin receptors (Saito et al.,Proc. Natl. Acad. Sci. USA 92: 10227-31 (1995); Pardridge et al., Pharm.Res. 12: 807-816 (1995); Broadwell et al., Exp. Neurol. 142: 47-65(1996)). This first antibody is covalently linked to an antibody whichbinds β-amyloid. Alternatively, coupling the β-amyloid antibodies toligands which bind these receptors (e.g., insulin, transferrin, or lowdensity lipoprotein) will also produce a vectorized antibody competentfor delivery to the brain from the circulation (Descamps et al., Am. J.Physiol. 270: H1149-H1158 (1996); Duffy et al., Brain Res. 420: 32-38(1987); Dehouck et al., J. Cell Biol. 138: 877-889 (1997)).

[0053] A vector moiety can be chemically attached to the anti-β-amyloidantibody to facilitate its delivery into the central nervous system.Alternatively, the moiety can be genetically engineered into theantibody as an integral component. This vector component can be forexample, an anti-transferrin receptor antibody or anti-insulin receptorantibody which binds the receptors present on the brain capillaryendothelial cells (Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-22(1993); Pardridge et al., J. Pharmacol. Exp. Ther. 259: 66-70 (1991);Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31(1995); Friden etal., J. Pharm. Exper. Ther. 278: 1491-1498 (1996)) which make up theblood-brain barrier. The resulting bifunctional antibody will attach tothe appropriate receptors on the luminal side of the vessel (Raso etal., J. Biol. Chem. 272: 27623-27628 (1997); Raso et al., J. Biol. Chem.272: 27618-27622 (1997); Raso, V. Anal. Biochem. 222: 297-304 (1994);Raso et al., Cancer Res. 41: 2073-2078 (1981); Raso et al., Monoclonalantibodies as cell targeted carriers of covalently and non-covalentlyattached toxins. In Receptor mediated targeting of drugs, vol. 82. G.Gregoriadis, G. Post, J. Senior and A. Trouet, editors. NATO AdvancedStudies Inst., New York. 119-138 (1984)). Once bound to the receptor,both components of the bispecific antibody pass across the blood-brainbarrier by the process of transcytosis. Anti-β-amyloid antibodies whichhave entered the brain interact directly with both β-amyloid plaques andthe soluble β-amyloid pool. It has been estimated that concentrations ofmacromolecules in the 10⁻⁸-10⁻⁷M range can be achieved in the brainusing vector-mediated delivery via these brain capillary enrichedprotein target sites (Maness et al., Life Sciences 55: 1643-1650 (1994);Lerner et al., Science 252: 659-667 (1991)). Importantly, the vectorappears safe since animals dosed daily for two weeks with ananti-transferrin receptor antibody displayed no loss of integrity of theblood-brain barrier, using a radioactive sucrose probe (Broadwell etal., Exp. Neurol. 142: 47-65 (1996)).

[0054] The Exemplification details the production of vectorizedbispecific antibodies which bind β-amyloid. The bispecific antibodiestranscytose across the blood brain barrier via a first specificity whichbinds the transferrin receptor. Use of antibodies which bind thetransferrin receptor for delivery of agents across the blood brainbarrier is described by Friden et al. in U.S. Pat. No. 5,182,107; No.5,154,924; No. 5,833,988; and No. 5,527,527; the contents of which areincorporated herein by reference.

[0055] Results from experiments presented in the Exemplification sectionwhich follows indicate that the produced bispecific antibodies retaintheir separate specificities and are delivered across the blood-brainbarrier into the brain parenchyma and brain capillaries of a live animalwhen administered intravenously.

[0056] Alternate methods for the production of bispecific antibodieshave been described for genetically engineering bispecific reagents orfor producing them intracellularly by fusing the two different hybridomaclones (Holliger et al., Proc. Natl. Acad. Sci. 90: 6444-6448 (1993);Milstein et al., Nature 305: 537 (1983); Mallander et al., J. Biol.Chem. 269: 199-206 (1994)). Vectorized bispecific antibodies produced bythese techniques can also be used in the methods of the presentinvention.

[0057] Since the introduction of whole antibodies into the brain mightbe detrimental if they were to fix complement and promotecomplement-mediated lysis of neuronal cells, it may be beneficial toproduce and utilize smaller vectorized F(ab′)₂ bispecific reagents. Ithas been shown that aggregated β-amyloid itself can fix complement inthe absence of any antibody and that the resulting inflammation maycontribute to the pathology of Alzheimer's disease. The possibility ofintracerebral antibody having a similar effect can be greatly reduced byeliminating the Fc region of the antibody. Moreover, since coupling ofFab′ halves uses the intrinsic hinge region cysteines, no extraneoussubstituent linkage groups need be added. Faster or more efficient entryinto the brain represents another potential advantage that smallerF(ab′)₂or Fv₂ reagents may provide for intracerebral delivery. Inaddition, the two types of vectorized molecules may have differentbiodistribution and plasma half-life characteristics (Spiegelberg etal., J. Exp. Med. 121: 323 (1965)).

[0058] Depending on their design, anti-β-amyloid bispecific antibodiesin the brain can reduce soluble β-amyloid and β-amyloid deposits bythree potential mechanisms. An anti-β-amyloid bispecific antibody thattightly binds soluble β-amyloid will not only sequester the peptide but,due to efflux of vectorized molecules from the central nervous system(Kang et al., J. Pharm. Exp. Ther. 269: 344-350 (1994)), may also carrythe bound β-amyloid out of the brain, releasing it into the bloodstream. Such a clearance mechanism would lead to a continuous cycling ofβ-amyloid out of the brain. In addition, if the antibodies havecatalytic activity, they will directly reduce the levels of harmfulβ-amyloid by degradation. Since catalytic antibodies exhibit turnover,each antibody can inactivate many β-amyloid molecules. Thus much lessvectorized bispecific antibody has to be delivered into the brain toachieve the desired depletion of β-amyloid.

[0059] To be effective the anti-β-amyloid sites of a bispecific antibodymust be empty before passage out of the blood and into the brain.Therefore the concentration of bispecific antibody in animals mustexceed the level of β-amyloid circulating in the blood. Calculationsperformed based upon known β-amyloid levels (Scheuner et al., NatureMed. 2: 864-870 (1996)) and a medium-range plasma level of bispecificantibody expected in a treated animal indicated 99.9% of the bispecificantibodies that enter the brain will have unoccupied anti-β-amyloidcombining sites.

[0060] Another way of delivering antibodies to the brain is via directinfusion of anti-β-amyloid antibodies into the brain of an animal. Thistechnique gives these antibodies immediate access to β-amyloid in thebrain without having to cross the blood-brain barrier. Direct infusioncan be accomplished via direct parenchymal or intracerebroventricularinfusion (Knopf et ale, J. Immunol. 161: 692-701 (1998)). Briefly, theanimal is anesthetized and placed in a stereotaxic frame. A midsagittalincision is made on the scalp to expose the skull and the underlyingfascia is scraped away. A hole is drilled to accept a sterilized lengthof stainless steel hypodermic tubing, which is stereotaxically advancedso that its tip is appropriately located in the brain. A guide cannulais then attached to the skull and sealed. The cannula remains in placefor multiple infusions of antibody into the brain. A bolus of a sterile50 mg/ml solution of a monoclonal anti-β-amyloid can be infused over a2-8 minute period into an immobilized animal via an injection cannula.

[0061] Delivery of catalytic antibodies into the brain of an animal viaone of the above described methods, can also be used to disaggregateamyloid plaques present in the brain. The advantage of delivering anβ-amyloid-specific catalytic antibody into the brain is two-fold. Theβ-amyloid peptide is permanently destroyed by such antibodies and, sincecatalysis is continuous, each antibody inactivates many target β-amyloidmolecules in the brain. Thus much less antibody has to be infused intothe central nervous system to achieve the desired depletion ofβ-amyloid.

[0062] The amount of antibody to be administered or delivered to theanimal should be sufficient to cause a significant reduction inβ-amyloid levels in the brain of the animal. The appropriate amount willdepend upon various parameters (e.g., the particular antibody used, thesize and metabolism of the animal, and the levels of endogenousβ-amyloid) and is to be determined on a case by case basis. Suchdetermination is within the means of one of average skill in the artthrough no more than routine experimentation.

[0063] It is expected that additional benefits with respect to loweringbrain β-amyloid levels and preventing or disaggregating amyloid plaquescan be achieved through utilizing a combination of one or more of theabove described approaches.

Exemplification

[0064] Section 1: Retention of β-amyloid in the Circulation

[0065] Synthesis of β-amyloid Peptide Antigens

[0066] The amino acid sequence of the 43 residue β-amyloid peptide (Aβ)is listed in FIG. 1. To determine which sites on this Aβ peptide werebest suited for antibody-mediated therapy, three key regions(amino-terminal, central and carboxy-terminal) of the Aβ 43-mer werechosen to generate epitope-specific vaccines. These shortened peptidesserved as antigenic epitopes to induce a highly specific antibodyresponse.

[0067] Monoclonal antibodies to the amino-terminal region of Aβ havebeen shown in the past to have the ability to solubilize Aβ aggregates(Solomon et al., Proc. Natl. Acad. Sci. USA 94(8): 4109 (1997); Solomonet al., Proc. Natl. Acad. Sci. USA 93(1): 452 (1996)). A peptideconsisting of the amino-terminal region of Aβ was similarly designed forthe present experiments (shown in FIG. 2 and listed in SEQ ID NO: 2) andused to elicit amino-terminal specific antibodies that bind Aβ. A Cysresidue was added to the C-terminus of the Aβ sequence to provide asuitable linkage group for coupling this peptide to an antigenic carrierprotein such as maleimide-activated Keyhole Limpet Hemocyanin (KLH).

[0068] A peptide encompassing the central region of Aβ was alsosynthesized (shown in FIG. 3 and listed in SEQ ID NO: 3). A Cys residuewas placed at the N-terminus of the Aβ sequence to provide a sulfhydryllinkage group for coupling the peptide to antigenic carrier proteinssuch as maleimide-activated KLH.

[0069] To produce an antigen for eliciting an immune response directedagainst the carboxy-terminus of Aβ (Suzuki et al., Science 264: 1336(1994)), a decapeptide encompassing the N-terminal region of Aβ, with anadditional Cys residue at the N-terminus, was synthesized (Shown in FIG.4, and listed in SEQ ID NO: 4). The Cys substitution was designed toprovide a sulfhydryl linkage group for coupling the peptide to antigeniccarrier proteins such as KLH.

[0070] Coupling the Peptides to an Antigenic Carrier Protein

[0071] The different Cys containing Aβ peptides were individuallythioether-linked to maleimide-activated KLH. A multivalent Aβ vaccinewas also produced by simultaneously linking all three of these peptidesto maleimide-activated KLH. In addition the full-length Aβ 43-mer waslinked to KLH using glutaraldehyde.

[0072] Antibodies Elicited with the β-amyloid Vaccines

[0073] Normal BALB/c mice were immunized by standard procedures with theKLH-linked Aβ vaccines described above. The mice were either bled orsacrificed for removal of the spleen for hybridoma production. Sera andmonoclonal antibodies obtained were characterized for binding to Aβ.

[0074] Table 1 shows the results from an ELISA run with {fraction(1/100)} diluted serum from two non-immunized control mice versus{fraction (1/100)} and {fraction (1/1000)} diluted serum from a mousethat was immunized with a central region Aβ peptide-KLH vaccine. Thefree Aβ peptide was adsorbed directly onto the microtitre plate to avoiddetection of anti-KLH antibodies in the serum. TABLE 1 ELISA for Bindingto the Central Region Aβ Peptide Antibody Bound Addition (O.D. 450 nm)Control Serum A 1/100 0.666 Control Serum B 1/100 0.527 Mouse 1antiserum 1/100 3.465 Mouse 1 antiserum  1/1000 2.764

[0075] Monoclonal antibodies raised against this central region Aβpeptide and produced by hybridoma fusion were identified using the abovedescribed ELISA assay. A binding assay was performed to determinewhether the monoclonal anti-Aβ antibodies identified also bound to thefull length Aβ peptides. ¹²⁵I-Aβ₁₋₄₃ probe was incubated with hybridomasecretions from the indicated clones. A standard polyethylene glycolseparation method was used to detect ¹²⁵I-Aβ₁₋₄₃bound antibody (Table2). Results presented in Table 2 indicate that the antibodies generatedto the peptide fragments also bound full length Aβ₁₋₄₃. TABLE 2¹²⁵I-Aβ₁₋₄₃ Binding Assay ¹²⁵I-Aβ₁₋₄₃ Bound Addition (cpm) Control Hy3,171 Control Hy 2,903 6E2 15,938 6E2 1/10 9,379 3B1 12,078 3B1 1/103,353 8E3 10,789 8E3 1/10 3,249

[0076] It was reported that when ¹²⁵I-Aβ₁₋₄₀ is added to human plasma,-89% binds to albumin (Biere et al., J. of Biol. Chem. 271(51): 32916(1996)). This raises the concern that the bound albumin will interferewith antibody binding. Binding assays were performed in the presence andabsence of serum albumin, to determine whether albumin bindinginterferes with antibody binding to Aβ. The ability of purified 5A11monoclonal anti-Aβ antibody to bind ¹²⁵I-Aβ₁₋₄₀ was unaffected by thepresence of human serum albumin (HSA) at 60 mg/ml, even though this wasa 500-fold molar excess over the antibody concentration (Table 3). Theseresults indicate that the ability of antibodies to bind to and sequesterAβ in the blood will not be attenuated by the presence of other bindingproteins. TABLE 3 ¹²⁵I-Aβ₁₋₄₀ Binding to Antibody in the Presence ofHuman Serum Albumin* ¹²⁵I-Aβ₁₋₄₀ Bound Specifically Bound Addition (cpm)(% of total added) Control 8,560 — + 5A11 anti-Aβ 64,589 79 Control +HSA* 3,102 — + 5A11 anti-Aβ + HSA* 55,304 75

[0077] Monoclonal Antibody Production

[0078] A mouse was immunized with a KLH conjugate of the central regionAβ₁₀₋₂₅ peptide (This peptide antigen had a phenylalanine statinetransition state analog at an amide linkage, discussed further inSection II, below). A hybridoma fusion was performed and the resultingmonoclonal antibodies analyzed to characterize the specificity of theimmune response to the vaccine. Hybridoma supernatants produced in thefusion were screened using ELISA to assess their binding to the Aβ₁₋₄₃peptide.

[0079] The monoclonal antibodies produced were determined to bind to theAβ₁₋₄₃ peptide adsorbed directly onto an ELISA plate. Strong colorreactions were obtained in this ELISA using only 10 μl of hybridomasupernatant while the addition of media alone produced low backgroundcolor. These results indicate that the antibodies not only bound to thesmall peptide immunogen but they were also reactive with the full-lengthAβ₁₋₄₃. Importantly, antibodies bound to the carrier-free Aβ peptideadsorbed directly onto microtitre plates, showing their specificity forthe peptide rather than the immunogenic carrier. The high affinity 5A11monoclonal antibody (Table 3) was obtained from this hybridoma fusion.

[0080] A second mouse was immunized with a KLH conjugate of the Aβ₃₅₋₄₃analog encompassing the C-terminal region of Aβ. Serum from the mousewas screened for reaction with Aβ₁₋₄₃ adsorbed directly onto the ELISAwells. The assay results are presented in Table 4. The spleen of thismouse was then used for a hybridoma fusion to further characterize thespecificity of its immune response. Importantly, none of the miceimmunized with Aβ vaccines or the anti-Aβ ascites-producing micedisplayed ill effects even though some of those induced antibodiescross-react with mouse Aβ and mouse amyloid precursor protein. TABLE 4ELISA for Binding of Antiserum Directed to the Carboxy-terminal AβPeptide Antibody Bound (O.D. 450 nm) Addition Native Aβ₁₋₄₃ ControlSerum 0.484 Mouse Antiserum 1.765

[0081] Monoclonal antibodies from hybridoma clones generated above werescreened for binding to the small carboxy-terminal peptide Aβ₃₅₋₄₃ andthe full-length Aβ₁₋₄₃. Results are presented in FIG. 5. The monoclonalantibodies bound to the carboxy-terminal locus on each of thesecarrier-free Aβ peptides adsorbed directly to the microtitre plate,confirming their specificity for the peptide rather than the immunogeniccarrier. The clones were also tested with Aβ₁₋₄₀ to identify antibodieswhich do not react with this shortened, 40 amino acid residue version ofAβ and thus will specifically bind to the carboxy-terminus of Aβ₁₋₄₃(FIG. 5). Used therapeutically, this vaccine should elicit antibodieswhich will preferentially bind the less abundant, but more noxiousAβ₁₋₄₃ species in the blood as opposed to the smaller and lessdetrimental Aβ₁₋₄₀.

[0082] In a separate experiment, mice were immunized with a vaccinecomprised of a cocktail of the three distinct KLH-peptide antigens(FIGS. 2-4) representing the distinct regions of β-amyloid (FIG. 1).Control mice were immunized with KLH alone. The antigens were emulsifiedin complete Freunds adjuvant prior to the first injection and inincomplete Freunds adjuvant for subsequent injections. Tests wereperformed on diluted serum from these Aβ-KLH immunized mice to determinethe presence of specific anti-Aβ antibodies. The Aβ₁₋₁₆, Aβ₁₄₋₂₅,Aβ₃₄₋₄₃, Aβ₁₋₄₀, and Aβ₁₋₄₃ peptides were used to identify antibodyspecificity. The peptides were adsorbed directly onto an ELISA plate.The results are presented in Table 5. The results indicate that miceimmunized with the cocktail of the three peptide antigens produced serumcontaining antibodies which react with the amino-terminal, centralregion, and carboxyl-terminal peptides, as well as with the full-lengthAβ 40-mer and 43-mer. The constant presence of this spectrum of anti-Aβantibodies will be very effective in binding all of the soluble Aβ inthe peripheral circulation of a vaccinated animal. TABLE 5 ELISA toMeasure the Serum Antibodies Present in Immunized Mice ELISA READING(O.D. 450 nm) Immunogen Aβ₁₋₁₆ Aβ₁₄₋₂₅ Aβ₃₄₋₄₃ Aβ₁₋₄₀ Aβ₁₋₄₃ Mouse 1(Control) KLH 0.076 0.038 0.064 0.042 0.066 Mouse 2 Aβ-KLH 3.013 1.2583.191 2.337 2.598 Cocktail Mouse 3 Aβ-KLH 1.484 1.180 2.068 1.758 1.680Cocktail Mouse 4 Aβ-KLH 1.486 1.072 2.276 1.444 1.709 Cocktail

[0083] Vaccine Trials in Non-human Primates

[0084] Given the potential importance of β-amyloid vaccine therapy forhuman patients of Alzheimer's disease, a human-compatible, alum-based Aβpeptide vaccine preparation has been tested in non-human primates.Antibody production and safety studies for the human-compatibleβ-amyloid vaccines have commenced in Cynomolgus monkeys (Macacafascicularis). This animal system is highly relevant to humanapplications since the predicted amino acid sequence of β-amyloid inthese primates is identical to humans, and their basic physiology andimmunological systems closely approximate those which will beencountered in a clinical situation. Cynomolgus monkeys were vaccinatedmonthly and were periodically bled to monitor anti-Aβ levels in theserum. The monkeys were also observed for any ill effects.

[0085] The Cynomolgus monkeys mounted a strong immune response to asingle injection of the simplest vaccine preparation composed of thefull length β-amyloid peptide adsorbed to an aluminum hydroxide gel. Thespecificity of those early anti-β-amyloid antibodies was characterizedby ELISA using various Aβ peptide fragments (Table 6). This analysisindicated that the monkeys produced antibodies that bind to thefull-length peptide and react with its amino-terminal, central andcarboxyl-terminal regions. TABLE 6 ELISA to Measure the Serum AntibodiesPresent in Aβ Vaccinated Macaca fascicularis ELISA READING (O.D. 450 nm)Vaccination Schedule Aβ₁₋₁₆ Aβ₁₄₋₂₅ Aβ₃₄₋₄₃ Aβ₁₋₄₀ Aβ₁₋₄₃Pre-Vaccination 0.511 0.404 0.370 0.380 0.235 Aβ/Alum (1st month) 2.1151.687 0.671 2.393 2.479

[0086] Importantly, the vaccinated monkeys are perfectly healthy andappear compatible with the anti-Aβ antibodies that have been circulatingin their body for over three months. Thus far, there are no apparentside effects due to cross-reaction of the anti-Aβ antibodies withnaturally occurring β-amyloid precursor protein or other vitalcomponents. These animals were closely observed by a veterinarian, andhave exhibited no signs of autoimmune disease, immune complex disease orany other adverse/toxic reaction to the vaccination.

[0087] In continuing experiments boost injections will be performed asper usual methods The sera produced will be monitored for antibodyspecificity and affinity parameters as the immune response intensifiesand matures. At termination, a complete necropsy and histopathologicalexamination will be performed on the monkeys. Genetically engineered Aβvaccines, discussed below, will also be evaluated in the Cynomolgusmonkeys to determine if they will prove to be even better immunogens.

[0088] Antibodies Affect the Distribution of ¹²⁵I-Aβ in Normal Mice

[0089] Anti-Aβ antibodies in the circulation cannot cross theblood-brain barrier to a significant extent and therefore should act asa sink that prevents ¹²⁵Aβ₁₋₄₀ from reaching the brain. This retentioneffect was demonstrated by measuring the blood levels in mice 4 h afterinjecting them with equal amounts of ¹²⁵I-Aβ₁₋₄₀ either alone or alongwith our 5A11 anti-Aβ monoclonal antibody (Table 7). The passage of¹²⁵I-Aβ₁₋₄₀ out of the peripheral circulation was greatly curtailed inanimals which concomitantly received the specific anti-Aβ antibody. Thatfinding extends the in vitro results obtained with the 5A11 antibody(Table 3) by demonstrating the antibody can effectively bind Aβ in anexperimental animal. The observation that animals treated with thisantibody retained 10-times more ¹²⁵I-Aβ₁₋₄₀ in the circulation indicatesthat the equilibrium distribution of Aβ in the body can be dramaticallyaltered by selective sequestration in the blood. TABLE 7 Anti-AβAntibody Impedes the Passage of ¹²⁵I-Aβ₁₋₄₀ Out of the Circulation¹²⁵I-Aβ₁₋₄₀ in Blood Mouse Injected With; (cpm/gm) ¹²⁵I-Aβ₁₋₄₀ alone27,300 ¹²⁵I-Aβ₁₋₄₀ + 5A11 anti-Aβ 278,900

[0090] Genetically Engineered Vaccines

[0091] Genetically engineered β-amyloid antigen vaccines for use inhumans are currently being developed in order to induce protectivelevels of anti-β-amyloid antibodies. β-amyloid fragments will beengineered into chimeric Aβ vaccines which incorporate highlyimmunogenic carrier moieties to increase the appropriate antigenicresponse in a human patient. Carrier moieties suitable for use includediphtheria toxoid (DT) and the hepatitis B core antigen (HBcAg). Theserepresent powerful delivery systems for β-amyloid peptides, and areknown to induce an excellent, high titer immune response when used withalum as an adjuvant.

[0092] DT is licensed for use as a conjugate vaccine for H. influenzaetype B and renders this immunogen T-cell dependent. The expression of DTin recombinant E. coli is high. One or more of the above describedβ-amyloid peptides will be fused at the C-terminus of the catalyticdomain of DT, or at either end of the combined transmembrane/receptorbinding domains of DT. The produced fusions will be used with analuminum hydroxide gel adjuvant to generate potent vaccines.

[0093] High titers of antibody directed against heterologous epitopeshave been produced using the HBcAg delivery systems and aluminumhydroxide gel adjuvant. HBcAg has several distinct advantages as afusion partner for Aβ peptides. The immunodominant internal site betweenamino acids 75 and 81 can accommodate heterologous sequences up to 45amino acids. The core self-assembles into larger 27 nm particles thatare highly immunogenic. Furthermore, HBcAg can be produced inrecombinant E. coli at elevated levels.

[0094] The genetically engineered vaccines produced will be tested foreffectiveness in depleting or preventing plaques using mouse and otherrelevant animal models. Antibody production and safety trials for thevaccines will be conducted in Cynomolgus monkeys.

METHODS OF THE INVENTION

[0095] Peptide Synthesis.

[0096] The 40mer Aβ₁₋₄₀, the 43mer Aβ₁₋₄₃, and the three small Aβpeptides Aβ₁₋₁₆, Aβ₁₀₋₂₅, and Aβ₃₅₋₄₃, were synthesized by standardautomated Fmoc chemistry. Newly synthesized peptides were purified byHPLC and their composition was verified by mass spectral and amino acidanalysis. The Aβ 43mer was obtained from a commercial source (Bachem,Torrance, Calif.).

[0097] Conjugation of β-amyloid Peptides to Immunogenic Carriers.

[0098] The small Aβ peptides were linked to the KLH carrier protein inorder to render them antigenic. A Cys residue was strategically placedat the N- or C-terminal end of these Aβ peptides to provide a suitablelinkage group for coupling them via a thioether bond to maleimideactivated carrier proteins. This linkage is stable and attaches thepeptide in a defined orientation. Addition of ˜20 peptides/KLH istypically obtained by this conjugation method. The longer, full lengthAβ peptides were linked to carrier proteins using a glutaraldehydecoupling procedure.

[0099] β-amyloid Antigen Cocktail.

[0100] The three Aβ peptides shown in FIGS. 2-4 were each individuallyconjugated to KLH. 20 μg of each of these three conjugates was thenmixed together. This mixture was emulsified with complete Freundsadjuvant and injected i.p. into mice. Subsequent monthly i.p. boosterinjections used the same cocktail mixture emulsified in incompleteFreunds adjuvant. Control mice received a similar immunization protocolbut using KLH which had not been conjugated with the Aβ peptides.

[0101] Immunization of Mice.

[0102] Normal BALB/c mice were immunized by standard procedures with theKLH-linked Aβ vaccines described above. Briefly, mice were injected i.p.with antigen emulsified in complete Freunds adjuvant, followed by asecond course in incomplete Freunds adjuvant. The mice were i.v. boostedwith antigen in PBS three days prior to bleeding them or removing thespleen for hybridoma fusions to produce monoclonal antibodies.

[0103] None of the mice immunized with Aβ vaccines or the anti-Aβascites-producing mice displayed ill effects even though some of theantibodies cross-reacted with mouse Aβ and mouse amyloid precursorprotein.

[0104] ELISA.

[0105] The presence of bound anti-peptide antibodies was revealed byusing a peroxidase-labeled anti-mouse IgG probe followed by thechromogenic substrate (Engvall et al., Immunochemistry 8: 871-875(1971)).

[0106] Binding Assay.

[0107] Both Aβ₁₋₄₃ and Aβ₁₋₄₀ were radiolabeled with ¹²⁵I. The iodinatedpeptide was separated from unlabeled material by HPLC to giveessentially quantitative specific activity (˜2000 Ci/mmol) (Maggio etal., Proc. Natl. Acad. Sci. 89: 5462 (1992)). ¹²⁵I-Aβ₁₋₄₃ probe wasincubated for 1 h at 23° C. with Hy media taken from hybridoma clonesproducing monoclonal anti-Aβ antibodies. A standard polyethylene glycolseparation method was used to detect the amount of ¹²⁵I-Aβ₁₋₄₃ bound toantibody.

[0108] β-amyloid Vaccines for Primates.

[0109] The immunogen used was a sytheticAβ peptide encompassing aminoacids 1-41 of the Aβ protein. This peptide was purified by HPLC andfreeze-dryed and then resuspended in sterile water at a concentration of1.5 mg/ml. The vaccine was prepared by mixing 7.5 ml of a 2% aluminumhydroxide gel adjuvant (Alhydrogel, Superfos Biosector, Denmark),referred to herein as alum gel, with 7.5 ml of the peptide. Tests showedthat all of the peptide was adsorbed to the alum gel after mixing for 12hours at 25° C.

[0110] Monkeys were initially vaccinated by intramuscular (i.m.)injection of 0.5 ml of the alum-adsorbed peptide. A second vaccination(boost) of the same vaccine preparation (0.5 ml) was administered amonth later. Subsequent identical monthly injections (boosts) will begiven until the experiment is terminated.

[0111] Genetically Engineered Vaccines.

[0112] Highly immunogenic carrier moieties will be used to constructchimeric Aβ vaccines. Moieties used will include diphtheria toxoid (DT)and the hepatitis B core antigen (HBcAg). The HBcAg expression systemwill be utilized (Schodel et al., Infect. and Immun. 57: 1347-1350(1989); Schodel et al., J. of Exper. Med. 180: 1037-1046 (1994); Schodelet al., J. of Virology 66: 106-114 (1992); Milich et al., Annals NewYork Academy of Sciences: 187-201 (1993)). The amino terminal end of thecatalytic domain of HBcAg has a signal sequence which should allow theAβ fusion protein to be secreted into the culture medium. The culturemedium will be concentrated using a large Amicon ultrafiltration device,and the concentrate then chromatographed on a large Superdex 75 column.Recombinant products obtained from within lysed cells will be separatedfrom bacterial protein using a combination of anion exchange and sizeexclusion FPLC.

[0113] Section II: Eliciting Monoclonal Antibodies with Transition StateAntigens

[0114] Transition State Peptide Antigens

[0115] Different types of transition state peptide antigens weresynthesized to use in the generation of antibodies which preferentiallyrecognize (hydrolysis) transition states of Aβ at a predetermined amidelinkage position.

[0116] A series of statine (Sta) transition state analogs encompassingthe carboxy-terminal region of Aβ(Cys-Met-Val-Gly-Gly-Val/Sta-Val/Sta-Ile/Sta-Ala-Thr) were synthesized.Replacement of the proposed scissile peptide linkage between Val₃₉ andVal₄₀, Val₄₀ and Ile₄₁, and Ile₄₁ and Ala₄₂, with a “statyl” moiety(—CHOH—CH₂—CO—NH—) was designed to elicit catalytic antibodies thathydrolytically cleave Aβ at one of these sites (FIG. 6). A Cys residuewas placed at the N-terminal position of these peptides to provide asuitable linkage group for coupling to a maleimide-activated carrierprotein.

[0117] A series of phenylalanine statine (PhSta) transition stateanalogs encompassing the central region of Aβ(Cys-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe/PhSta-Phe/PhSta-Ala-Glu-Asp-Val-Gly-amide)was synthesized in this laboratory.

[0118] Replacement of the proposed scissile peptide linkage betweenPhe₁₉ and Phe₂₀, and between Phe₂₀ and Ala₂₁, with a statyl moiety(—CHOH—CH₂—CO—NH—) was designed to elicit catalytic antibodies thathydrolytically cleave Aβ at these sites (FIG. 7). A Cys residue wasplaced at the C-terminus of these peptides to provide a sulfhydryllinkage group for coupling the peptides to antigenic,maleimide-activated carrier proteins such as KLH.

[0119] A structural comparison (FIG. 8) was made between the native Aβpeptide and the transition state phenylalanine statine Aβ peptide usinga graphics workstation. An energy minimization algorithm (2000iterations) was applied to arrange each peptide in its most favorableconformation.

[0120] The peptide link (—CO—NH—) between Phe₁₉ and Phe₂₀was replacedwith an elongated “statyl” moiety (—CHOH—CH₂—CO—NH—) and an energyminimization was applied. This orientation shows the difference betweenthe planar peptide link (—CO—NH—) of natural Aβ (left) versus theextended, tetrahedral “statyl” moiety (—CHOH—CH₂—CO—NH—) in thetransition state peptide (right).

[0121] An antibody combining site complementary to a tetrahedral statinetransition state analog will force the planar peptide bond of the Aβsubstrate into a transition state-like conformation. Such distortionshould catalyze the cleavage of Aβ at that locus in the peptidesequence.

[0122] The possibility of using a reduced peptide bond linkage to mimicthe transition state during hydrolysis of an amide linkage was alsoexplored. A reduced peptide bond linkage can be easily placed at almostany site in the Aβ molecule to produce a reduced peptide bond transitionstate analog. This analog can also be used to elicit catalyticantibodies that will hydrolytically cleave Aβ at the chosen site. Thereduced peptide bond transition state Aβ analog made was the(Gln-Lys-Leu-Val-Phe-CH₂—NH₂ ⁺-Phe-Ala-Glu-Asp-Val-Gly-Cys-amide)central region peptide; [calculated 1,342 (M+1); observed 1,344].

[0123] A structural comparison (FIG. 9) was made between the native Aβpeptide and the reduced peptide bond transition state Aβ analog using agraphics workstation. The peptide link (—CO—NH—) between Phe₁₉ and Phe₂₀was replaced with a reduced peptide bond (—CH₂—NH₂ ⁺—) and an energyminimization was applied. The orientation shown indicates the differencebetween the planar peptide link (—CO—NH—) of natural Aβ (left) versusthe corresponding tetrahedral moiety (—CH₂—NH₂ ⁺—) in the reducedpeptide bond transition state analog (right). An energy minimizationalgorithm (2000 iterations) was applied to arrange each peptide in itsmost favorable conformation.

[0124] A phosphonamidate transition state analog of the carboxy-terminalregion of Aβ has also been synthesized (FIG. 10). Replacement of theproposed scissile peptide linkage between Gly₃₈ and Val₃₉ with aphosphonamidate moiety (—PO₂ ⁻—NH—) was designed to elicit catalyticantibodies that will hydrolytically cleave Aβ at this site. TheN-acetyl-Cys residue was placed at the position of Leu₃₄ to provide asuitable linkage group for coupling this peptide to an antigenic carrierprotein. The structures in FIG. 11 represent the putative transitionstate for peptide hydrolysis by zinc peptidases, versus structure of andthe phosphonate and phosphonamidate mimics. Similar tetrahedraltransition state intermediates are known to be formed by reaction witheach of the four classes of proteolytic enzymes, the serine-, cysteine-,aspartic- and metallo-peptidases.

[0125] A structural comparison was made between the native Aβ peptideand the transition state phosphonamidate Aβ peptide (FIG. 12) using agraphics workstation. The peptide link (—CO—NH—) between Gly₃₈ and Val₃₉was replaced with a phosphonamidate bond (—PO₂ ⁻—NH—) and an energyminimization was applied. The orientation shown in FIG. 12 illustratesthe difference between the planar peptide link (—CO—NH—) of native Aβ(left) versus the corresponding tetrahedral phosphonamidate bond (—PO₂⁻—NH—) in the transition state peptide (right).

[0126] An antibody combining site complementary to the tetrahedraltransition state analog on the right of FIG. 12, will force the normallyplanar bond of the Aβ substrate peptide on the left into a transitionstate-like conformation. Such bond distortion is expected to catalyzethe hydrolytic cleavage of the Aβ peptide at the Gly₃₈-Val₃₉ linkage.

[0127] Immunization with Transition State Peptide Antigens

[0128] Peptide antigens were coupled to the immunogenic carrier KLHprior to immunization of mice. Standard protocols were used to immunizeBALB/c mice with the KLH-linked Aβ peptides described in the precedingsections. Briefly this procedure used i.p. injection of the differentantigens emulsified in complete Freunds adjuvant, followed by a secondcourse in incomplete Freunds adjuvant. Three days prior to hybridomafusion, the BALB/c mice were boosted i.v. with antigen in PBS.

[0129] A hybridoma fusion was performed using the spleen of a mouseimmunized with either a mixture of the phenylalanine statine transitionstate antigens generated (FIG. 7), a mixture of the statine (Sta)transition state Aβ antigens generated (FIG. 6), the reduced peptidebond transition state Aβ antigen generated (transition state mimiclocated between Phe₁₉-Phe₂₀), or the phosphoamidate transition state Aβantigen generated (transition state mimic located between Gly₃₈-Val₃₉).Monoclonal antibodies listed in Table 8 were generated from these mice.TABLE 8 Potential Cleavage Antibodies Analog Used Bonds Modified SitesGenerated statine Val₃₉-Val₄₀ Val₃₉-Val₄₀ 2B2, 2H6, 3F2, 4D3, 6A6, 1E4,Val₄₀-Ile₄₁   Val₄₀-Ile₄₁   11E9, 9D6, 5C7, 7C7, 1D12  Ile₄₁-Ala₄₂ Ile₄₁-Ala₄₂ phenylalanine- Phe₁₉-Phe₂₀ Phe₁₉-Phe₂₀ 6E2, 5A11, 6F11,2E3, 8E3, 5G4, statine  Phe₂₀-Ala₂₁   Phe₂₀-Ala₂₁  4C7, 8D12, 2C12, 4G7,5C7, 3C1, 4H9, 8E6, 1H2, 3B1, 2H11 reduced Phe₁₉-Phe₂₀ Phe₁₉-Phe₂₀ 6E7,6F6 peptide bond phospho-  Glu₃₈-Val₃₉   Gly₃₈-Val₃₉  in progressamidate

[0130] Demonstration of Aβ Binding by Generated Antibodies

[0131] It was very important to demonstrate that the anti-Aβ andanti-transition state Aβ monoclonal antibodies bound to the naturalAβ₁₋₄₃peptide which they were designed to sequester or cleave. To dothis, Aβ₁₋₄₀ and Aβ₁₋₄₃ were radiolabeled with ¹²⁵I and the iodinatedpeptide was then separated from unlabeled material by HPLC. Probe wasincubated with either purified anti-Aβ antibodies or media taken fromhybridoma clones producing anti-Aβ antibodies. The amount of ¹²⁵I-Aβ₁₋₄₃bound to antibody was determined using a polyethylene glycol separationmethod. Results of the experiment are presented in Table 3.

[0132] The data in Table 3 demonstrate the ability of the purified 5A11monoclonal anti-Aβ antibody to bind a high percent of ¹²⁵I-Aβ₁₋₄₀. Thisbinding assay was used to screen clones and purified antibodies (Table3) for their ability to bind Aβ. Similar procedures can also serve asthe basis for a competitive displacement assay to measure the relativebinding strength of different unlabeled Aβ peptides. (Note: with veryefficient catalytic antibodies this binding assay may have to beperformed on ice to ensure that no cleavage of Aβ occurs during the 1 hincubation time.) The assay rapidly identified clones producing highaffinity anti-Aβ antibodies.

[0133] Monoclonal antibodies from hybridomas obtained using thephenylalanine statine transition state Aβ-KLH antigen were screened byELISA to assess their binding to both the normal Aβ₁₋₄₃ peptide and tothe phenylalanine statine transition state Aβ peptide. Two majorpatterns were found (FIG. 13).

[0134] One group of antibodies (the left portion of FIG. 13) bound tothe immunizing transition state peptide and cross-reacted strongly withthe native Aβ₁₋₄₃ peptide (each was adsorbed directly onto the ELISAplate). The second group (the right portion) showed a high bindingpreference for the phenylalanine statine transition state Aβ peptide andreacted minimally with native Aβ₁₋₄₃.

[0135] Strong color reactions were obtained in this ELISA using only 10μl of hybridoma supernatant while Hy media alone or PBS gave a lowbackground (FIG. 13). These results demonstrate that the comparativeELISA screen, although only a semi-quantitative measure of binding,provides a means for identifying monoclonal antibodies that are highlyselective for, and most reactive with, the transition state.Importantly, the experiment was performed with carrier-free Aβ peptidesadsorbed directly onto microtitre plates, indicating antibodyspecificity for Aβ peptide rather than carrier.

[0136] These findings indicate that several of the generated anti-Aβtransition state antibodies were unique. They bound to both thephenylalanine statine- and normal-Aβ peptides. Their selectiverecognition of the transition state and weaker cross-reaction withnative Aβ₁₋₄₃ however indicates that this binding interaction is verydifferent from that shown by conventional anti-native Aβ antibodies. Itfurther indicates that these new antibodies may be able to force thenative Aβ peptide into a conformation resembling the transition statefor hydrolytic cleavage. Importantly, some of the antibodies whichshowed only minimal binding to Aβ₁₋₄₃ in this ELISA, did displaycross-reactivity with the natural peptide using a highly sensitive¹²⁵I-Aβ₁₋₄₃ binding assay (Table 3).

[0137] ELISAs were also performed to investigate the binding ofanti-statine analog antibodies to both the normal Aβ₁₋₄₃ peptide and tothe statine transition state Aβ peptide (FIG. 14). The antibodies boundto the C-terminal locus on these carrier-free Aβ peptides (adsorbeddirectly to the microtitre plate) confirming their anti-peptidespecificity. Most of the antibodies preferentially recognized thestatine Aβ transition state, but cross-reacted with native Aβ₁₋₄₃. Thisindicates that these new antibodies are able to force the native Aβpeptide into a conformation resembling the transition state forhydrolytic cleavage of its C-terminal amino acids. Such cleavage ispredicted to convert Aβ₁₋₄₃ into potentially less harmful shorterpeptides, like Aβ₁₋₄₀ or Aβ₁₋₃₉.

[0138] Clone 11E9 had the strongest preference for the statine analogand may be the most likely to have catalytic activity (FIG. 14). Severalclones displayed no difference in their reactivity with the nativeversus statine transition state Aβ peptide. The clones were also testedwith Aβ₁₋₄₀ to identify antibodies which do not react with thisshortened, 40 amino acid version of Aβ (FIG. 14). Used therapeutically,such antibodies should preferentially bind/cleave the less abundant, butmore noxious Aβ₁₋₄₃ species in the blood as opposed to the smaller andless detrimental Aβ₁₋₄₀.

[0139] Solid Phase and TLC Aβ Proteolytic Assays

[0140] A solid phase ¹²⁵I-labeled Aβ assay was developed to screenanti-transition state antibody hybridoma supernatants for specificproteolytic activity. The peptideCys-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Tyr-amide (SEQ IDNO: 5) which encompasses amino acids 14-25 of Aβ was radiolabeled andcoupled to a thiol-reactive, iodoacetyl-Sepharose gel to form anirreversible linkage. The product was incubated with anti-transitionstate antibody and assayed for the progressive release of soluble¹²⁵I-peptide from the solid phase matrix. Release of radioactivity fromthe ¹²⁵I-Aβ-Sepharose was used to identify catalytic activity (FIG. 15).The assay was verified by the ability of several different proteases torapidly hydrolyze this Sepharose-linked Aβ substrate. The peptide wasreadily accessible to proteolytic cleavage as revealed by a release ofsoluble ¹²⁵I-peptide that increased with incubation time.

[0141] The results presented in FIG. 15 indicate that theantibody-containing media of several clones released ¹²⁵I peptide at agreater rate than other clones from this fusion or the PBS and Hy mediumcontrols. Large amounts of these antibodies can be obtained, purifiedand tested at higher concentrations to achieve much faster rates ofcleavage and to verify that the antibodies are acting in a catalyticmode using conventional enzyme kinetics. By changing the composition ofthe ¹²⁵I-peptide this same strategy can be used to assay antibodiesreactive with different regions of Aβ.

[0142] A thin layer chromatography-based autoradiography assay wasdevised to obtain more definitive evidence for antibody-mediatedcleavage of Aβ. Selected anti-phenylalanine statine Aβ transition stateclones were expanded and ascites production induced. The differentmonoclonal antibodies were isolated using protein A-Sepharose. Two¹²⁵I-labeled peptides, Aβ₁₋₄₀ and a 17-mer, encompassing amino acids9-25 of Aβ, were used to test for peptide cleavage. The antibodies wereadded to the ¹²⁵I-peptides, allowed to incubate and the reaction mixspotted onto polyamide thin layer sheets which were then developed indifferent solvents. The migration of ¹²⁵I-products was followed byexposing the sheet using a quantitative phosphoimager systemQuantitation of the different labeled peptide fragments producedindicated that addition of the antibodies to the Aβ peptides lead tosignificant break down of the Aβ peptides compared to the untreatedpeptides (PBS).

[0143] Disaggregation of β-amyloid by Monoclonal Antibodies

[0144] The self-aggregation of synthetic Aβ peptides has been shownpreviously to lead to microscopic structures resembling amyloid plaquesin the brain (Solomon et al., Proc. Natl. Acad. Sci. USA 94: 4109-12(1997); Solomon et al., Proc. Natl. Acad. Sci. USA 93: 452-5 (1996))which exhibit the same bright green fluorescence upon exposure tothioflavin T. These aggregates are very stable and usually require harshdetergents or strong acids to dissolve. However, it has beendemonstrated that the binding of certain anti-Aβ monoclonal antibodiescan effectively inhibit the initial aggregation of this peptide and alsodisaggregate preformed Aβ complexes (Solomon et al., Proc. Natl. Acad.Sci. USA 94: 4109-12 (1997); Solomon et al., Proc. Natl. Acad. Sci. USA93: 452-5 (1996)).

[0145] A radioactive assay was used to quickly screen the differentmonoclonal antibodies produced by the present experiments for an abilityto dissolve preformed Aβ aggregates, made with ¹²⁵I-labeled andunlabeled soluble Aβ peptide. An aliquot of the labeled aggregate wasincubated with either PBS, the 5A11 anti-Aβ antibody or an equal amountof an irrelevant mouse antibody (7D3, anti-human transferrin receptor),and the level of released radioactivity was subsequently measured (Table9). The Aβ-specific 5A11 antibody solubilized 80% of the Aβ aggregateswhile an equal amount of the control antibody had only a minor effect,suggesting that the equilibrium was displaced by antibody-mediatedbinding of soluble Aβ. TABLE 9 Solubilization of ¹²⁵I-Aβ₁₋₄₀ Aggregateby Monoclonal Anti-Aβ Antibody ¹²⁵I-Aβ₁₋₄₀ in Ppt. Amount SolubilizedAddition (cpm) (% of PBS Control) PBS control 3,420 — + 5A11 anti-Aβ 67680 + 7D3 anti-TfR 2,458 27

[0146] Production of Vectorized Anti-Aβ/Anti-receptor BispecificAntibodies

[0147] Anti-Aβ antibodies were linked to anti-transferrin receptorantibodies (anti-TfR) which served as vectors for delivery of theanti-Aβ antibodies into the brain. The 7D3 mouse monoclonal antibody wasused as the anti-TfR part of the construct. 7D3 is specific for thehuman receptor and selectively immunostains cortical capillaries innormal human brain tissue (Recht et al., J. Neurosurg. 72: 941-945(1990)). Antibody attachment to the receptor is not blocked by an excessof human transferrin. The epitope recognized by this antibody istherefore distant from the receptor-ligand binding site. Bispecificantibodies constructed with this 7D3 antibody and an anti-Aβ antibodyare predicted to be useful for therapy in patients with Alzheimer'sdisease.

[0148] For studies in mouse models of Alzheimer's disease an anti-mousetransferrin receptor monoclonal antibody produced in the rat wasobtained. This antibody also appears to recognize a transferrin receptorepitope which does not involve ligand binding. The antibody thereforehas no effect on cell proliferation when using murine lines.

[0149] A series of functional assays were performed after completion ofthe synthesis, purification and size analysis of theanti-Aβ/anti-transferrin receptor bispecific antibodies. The vectorizedbispecific antibody, composed of a rat monoclonal antibody directedagainst the mouse transferrin receptor plus the 5A11 mouse anti-Aβmonoclonal antibody, was tested for the ability to attach to transferrinreceptor bearing mouse cells. Both components of the bispecific antibodywere detected on the cell membrane by cytofluorimetry (FIG. 16) whenthis duplex was reacted with transferrin receptor positive mouse cellsand probed using either a rat IgG-specific or mouse IgG-specificfluorescent secondary antibody reagent.

[0150] The capacity of the hybrid reagent to bind ¹²⁵I-Aβ comparedfavorably with that of the parent anti-Aβ antibody (Table 10). TABLE 10¹²⁵I-Aβ Binding to Bispecific Antibody ¹²⁵I-Aβ₁₋₄₀ Bound Addition (cpm)Control 4,199 + anti-Aβ 23,301 + anti-Aβ/anti-receptor 22,850

[0151] To ensure that both of these binding activities resided on thebispecific antibody, transferrin receptor positive cells were treatedwith the hybrid reagent, unbound material was washed away, and then thecells with bound antibody was exposed to ¹²⁵ I-Aβ₁₋₄₀. After washingaway unbound Aβ, the cell-bound radioactivity was compared to controlcells which had been identically prepared except for omission ofpretreatment with bispecific antibody. The results are presented inTable 11, and verify the dual specificity of this bispecific antibody byclearly showing that it can simultaneously attach to the cell membraneand bind ¹²⁵I-Aβ₁₋₄₀. TABLE 11 Bispecific Antibody-Mediated Binding of¹²⁵I-Aβ to Receptor-Positive Cells Pretreatment of Cells ¹²⁵I-Aβ₁₋₄₀Bound (cpm) None 2,367 + anti-Aβ/anti-transferrin receptor 11,476

[0152] Transcytosis of Bispecific Antibody into the Brain

[0153] A rat monoclonal anti-mouse transferrin receptor antibody wascoupled to a mouse monoclonal antibody (obtained from American TypeCulture Collection (ATCC TIB 219), also designated R17 217.1.3 (Cell.Immunol. 83: 14-25 (1984)), so that the entry of this new vectorizedbispecific construct into brain could be monitored. The bispecificantibody was labeled with ¹²⁵I and injected i.v. into normal mice. Afterdifferent lengths of time the mice were sacrificed and the amount of¹²⁵,-bispecific antibody that crossed the blood-brain barrier andentered the brain was gauged by a mouse capillary depletion method(Friden et al., J. Pharm. Exper. Ther. 278: 1491-1498 (1996); Trigueroet al., J. Neurochem. 54: 1882-1888 (1990)).

[0154] The amount of vectorized bispecific antibody found in the brainparenchyma or brain capillary fractions was measured followingdifferential density centrifugation of the brain homogenate. Thesevalues were plotted as a function of time after i.v. injection (FIG.17). The time-dependent redistribution of radiolabeled bispecificantibody from the capillaries and into the parenchyma was consistentwith its passage across the cerebral endothelial blood-brain barrier(Joachim et al., Nature 341: 6239:226-30 (1989)). Even greateraccumulation in the parenchyma is expected to occur if the antibodiesattach to Aβ in the cerebral plaques of plaque-bearing mice.

[0155] Monitoring the Brain Distribution of Bispecific Antibody in LiveMice

[0156] The ability to follow the entry and accumulation of vectorizedbispecific antibodies in the brain of live mice would greatly assist inthe development of the intracerebral treatment of plaque-bearing mice.Such a development would enable time-course studies and would greatlyreduce problems with inter-mouse variability. Preliminary studies with¹²⁵I labeled bispecific antibodies were performed to determine ifimmunoscintigraphy was feasible in this system. As a first step, eitherthe radiolabeled vectorized bispecific antibody (¹²⁵I-R17/5A11) or anon-vectorized control bispecific antibody were administered to separatemice. Sequential brain images were accumulated at 1, 6, 24 and 48 hoursfollowing i.v. administration of the ¹²⁵1-labeled bispecific antibodyprobes. Although this technique suffered from a difficulty indetermining how much of the signal was due to the levels of blood-borneradioactivity circulating through the brain, significant distinctionswere noted in the brain of mice treated with the mouse transferrinreceptor reactive bispecific antibody versus those receiving the controlbispecific antibody. When the vectorized agent was used, brain levelsincreased between 1 and 6 hrs and then declined to a much lower level at24 and 48 hrs. Mice treated with the control displayed no increasebetween 1 and 6 hrs. The reason for decreased brain levels at 24 hrs andbeyond is not known but might be due to dehalogenation of the bispecificantibody probes so that free ¹²⁵I is released. Alternative methodsutilizing radioactive labels such as ¹¹¹In (Sheldon et al., Nucl. Med.Biol. 18: 519-526 (1991)) or ⁹⁹Tc (Texic et al., Nucl. Med. Biol. 22:451-457 (1995)) attached to the vectorized bispecific antibody can beutilized in future experiments if the use of iodine presents a technicalproblem. This imaging technology will be useful for determining ifsmaller vectorized bispecific antibodies (eg. F(ab′)₂) with differentphysical properties and an altered biodistribution will penetrate intothe brain more effectively.

[0157] F(ab′)₂ Heterodimers for Vector-mediated Transport into the Brain

[0158] The introduction of whole antibodies into the brain might bedetrimental if they were to fix complement and promotecomplement-mediated lysis of neuronal cells. The development of smallervectorized F(ab′)₂ bispecific reagents is expected to avoid thisproblem. It has been shown that aggregated Aβ itself can fix complementin the absence of any antibody and that the resulting inflammation maycontribute to the pathology of Alzheimer's disease. The possibility ofintracerebral antibody having a similar effect would be greatly reducedby eliminating the Fc region of the antibody. Moreover, since couplingof Fab′ halves uses the intrinsic hinge region cysteines, no extraneoussubstituent linkage groups need be added.

[0159] Faster or more efficient entry into the brain represents anotherpotential advantage that smaller F(ab′)₂ or Fv₂ reagents provide forintracerebral delivery. Such modified bispecific agents can be preparedand compared to full-sized hybrid antibodies for their relativeeffectiveness in reaching the brain, crossing the blood-brain barrier,and affecting Aβ plaque development, by the methods described herein. Itis important to note, however, that only minor differences were foundwhen the capacity of differently-sized anti-transferrin receptorbispecific reagents for delivering toxins into cells byreceptor-mediated endocytosis was compared (Raso et al., J. Biol. Chem.272: 27623-27628 (1997)). This observation might indicate that littlevariation will be seen for transcytosis across the brain capillaryendothelial cells which form the blood-brain barrier. At the very leasthowever one would expect the two types of vectorized molecules to havedifferent biodistribution and plasma half-life characteristics(Spiegelberg et al., J. Exp. Med. 121: 323 (1965)).

METHODS OF THE INVENTION

[0160] Antigen Synthesis.

[0161] The statine and phenylalanine statine transition state peptideswere synthesized using automated Fmoc chemistry. Fmoc-statine (Sta),[N-Fmoc-(3S,4S)-4-amino-3-hydroxy-6-methyl heptanoic acid] andFmoc-“phenylalanine statine” (PhSta),[N-Fmoc-(3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid] were purchasedcommercially Each peptide was tested for purity by HPLC and itscomposition was verified by mass spectral and amino acid analysis.

[0162] The design strategy and methods for synthesizing phosphonamidate-and phosphonate-based transition state peptides are straightforward(Bartlett et al., Am. Chem. Society 22: 4618-4624 (1983); Bartlett etal., Biochemistry 26: 8553-8561 (1987)). The N-terminal portion of thepeptide (N-acetyl-Cys-Met-Val-Gly) was made using standard automatedFmoc chemistry. After cleavage from the resin the N-acetyl tetrapeptidewas treated with pyridine disulfide to protect its sulfhydryl group. Anacid chloride of Cbz-glycine phosphonate monomethyl ester (Bartlett etal., Am. Chem. Society 22: 4618-4624 (1983); Bartlett et al.,Biochemistry 26: 8553-8561 (1987)) was coupled withVal-Val-Ile-Ala-amide which was synthesized by automated Fmoc chemistry.The last amino acid of Aβ, Thr, was omitted due to potential problemswith its unprotected hydroxyl group. The product, Cbz-Gly-PO₂⁻—NH-Val-Val-Ile-Ala-amide has a phosphonamidate (methyl ester) bondbetween the Gly and Val residues. Next, the Cbz blocking group wasremoved using hydrogen so that the protected N-acetyl-Cys-Met-Val-Glypeptide could be added to the amino terminal end of this transitionstate peptide by HBTU-activated peptide linkage. Treatment withmercaptoethanol and rabbit liver esterase was used to deblock thepeptide. Each key component in the synthetic scheme was tested forpurity by HPLC and its composition was verified by mass spectral andamino acid analysis.

[0163] A reduced peptide bond linkage was placed at the indicated sitesin the Aβ molecule. Automated Fmoc chemistry was used to begin synthesisof the peptide. A pre-synthesized Fmoc amino aldehyde was then addedmanually and after the imide was reduced, automated synthesis wasresumed (Meyer et al., J. Med. Chem. 38: 3462-3468 (1995)).

[0164] Coupling of Antigen to Carrier.

[0165] The native and transition state Aβ peptides were coupled tomaleimide-activated KLH by standard procedures (Partis et al., J. Pro.Chem. 2: 263-277 (1983)), in order to elicit an immune response. A Cysresidue was strategically placed at the N- or C-terminal end of thepeptides to provide a suitable linkage group for coupling them via athioether bond to maleimide activated carrier proteins. This stablelinkage attaches the peptide in a defined orientation. Addition of ˜20peptides/KLH has been obtained based upon the transition state aminoacid content as determined by amino acid analysis of the hydrolyzedconjugates (Tsao et al., Anal. Biochem. 197: 137-142 (1991)).

[0166] Immunization of Mice.

[0167] Standard protocols were used to immunize mice with the KLH-linkedAβ peptides described in the preceding sections. Briefly this procedureused i.p. injection of the different antigens emulsified in completeFreunds adjuvant, followed by a second course in incomplete Freundsadjuvant. Three days prior to the hybridoma fusion, the BALB/c mice wereboosted i.v. with antigen in PBS. After 1 month animals were given aboost i.p. using the antigen emulsified with incomplete adjuvant. Serumfrom these animals was analyzed for anti-peptide antibodies by ELISA.BALB/c mice showing abundant antibody production were boosted by an i.v.injection with antigen and three days later they were used to generatehybridoma clones that secrete monoclonal antibodies.

[0168] None of the mice immunized with Aβ vaccines or the anti-Aβascites-producing mice displayed ill effects even though some of thoseinduced antibodies cross-react with mouse Aβ and mouse amyloid precursorprotein.

[0169] Hybridoma Production I.

[0170] A hybridoma fusion was performed using the spleen of a mouseimmunized with the phenylalanine statine transition state Aβ-KLHantigen. Spleen cells from mice with the highest titre were fused withmouse myeloma NS-1 cells to establish hybridomas according to standardprocedures (Köhler et al., Nature 256: 495 (1975); R. H. Kennett, FusionProtocols. Monoclonal Antibodies, eds. R. H. Kennett, T. J. McKearn andK. B. Bechtol. Plenum Press, New York. 365-367 pp. (1980)).

[0171]¹²⁵I-Aβ Binding Assay.

[0172] Aβ₁₋₄₀ and Aβ₁₋₄₃ were radiolabeled with ¹²⁵I and the iodinatedpeptide then separated from unlabeled material by HPLC to givequantitative specific activity (˜2000 Ci/mmol) (Maggio et al., Proc.Natl. Acad. Sci. 89: 5462-5466 (1992)). This probe was incubated for 1hat 23° C. with either purified anti-Aβ antibodies or media taken fromhybridoma clones producing anti-Aβ antibodies. A polyethylene glycolseparation method was used to detect the amount of ¹²⁵I-Aβ₁₋₄₃ bound toantibody. By using serial dilution, this assay can provide relativebinding affinities for the different hybridoma supernatants or purifiedantibodies.

[0173] Solid Phase Aβ Proteolytic Assay.

[0174] A solid phase ¹²⁵I-labeled Aβ assay was developed to screenanti-transition state antibody hybridoma supernatants for specificproteolytic activity. TheCys-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Tyr-amide peptide(SEQ ID NO: 5) encompassing amino acids 14-25 of Aβ was radiolabeledwith ¹²⁵I and the iodinated peptide was then separated from unlabeledmaterial by HPLC. The highly radioactive Aβ peptide was coupled to athiol-reactive, iodoacetyl-Sepharose gel to form an irreversible linkageAntibodies were added to the labeled Aβ, which was then assayed forprogressive release of soluble ¹²⁵I-peptide from the solid phase matrixat pH 7, 25° C. This assay was verified by the ability of severaldifferent proteases in to rapidly hydrolyze this Sepharose-linked Aβsubstrate Release of soluble ¹²⁵I-peptide increased with incubationtime.

[0175] Although Aβ is cleaved by several naturally occurring proteases,preliminary tests indicated that interference from high levels ofbackground hydrolysis was not a problem when assaying hybridomasupernatants of clones that did produce catalytic antibodies. A furtherprecaution that can be taken against exogenous proteases is carrying outall hybridoma cell fusions and cell culturing in serum-free media.

[0176] TLC Aβ Proteolytic Assay.

[0177] A thin layer chromatography-based autoradiography assay was usedto obtain more definitive evidence for antibody-mediated cleavage of Aβ.Selected anti-phenylalanine statine Aβ transition state clones wereexpanded and ascites production induced. The different monoclonalantibodies were isolated using protein A-Sepharose. The cleavage assayused ¹²⁵I-Aβ₁₋₄₀ and an ¹²⁵I-labeled 17-mer, encompassing amino acids9-25 of Aβ. Binding of the two ¹²⁵I-labeled peptides to the purifiedmonoclonal antibodies 5A11 and 6E2 was examined using either a PEGprecipitation assay or by a co-electrophoresis method Peptide cleavagewas tested by adding the antibodies to the ¹²⁵I-peptides, incubating andthen spotting the reaction mix onto polyamide thin layer sheets. Thechromatographs were developed in different solvents (eg. 0.5 N HCl, 0.5N NaOH or pH 7 phosphate buffer) and the migration of ¹²⁵I-products wasfollowed by exposing the sheet using a quantitative phosphoimagersystem.

[0178] Screen and Isolate Select Anti-Aβ Antibodies.

[0179] An ELISA was used to initially screen for anti-Aβ andanti-transition state Aβ peptide monoclonal antibodies. Both thetransition state peptide and the corresponding natural Aβ peptide wereadsorbed onto separate microtitre plates. The hybridoma supernatantswere screened using two assays so that the relative binding to bothnative and transition state Aβ peptides could be quantitated. Clonesproducing monoclonal antibodies that preferentially recognized thetransition state or bound Aβ with high affinity were selected forexpansion and further study.

[0180] Propagation and Purification of Monoclonal Antibodies.

[0181] Selected clones producing anti-Aβ antibodies and clones producinganti-receptor antibodies were injected into separate pristane-primedmice. Ascites were collected and the specific monoclonal antibodiesisolated. Purification of antibodies from ascites was accomplished usinga Protein A column or alternatively, antibodies were isolated fromascites fluid by (NH₄)₂SO₄ precipitation and passage over an S-300column to obtain the 150 kDa immunoglobulin fraction. Monovalent Fabfragments were prepared and isolated by established methods. Theirpurity was evaluated by SDS-PAGE under reducing and non-reducingconditions. 50-100 mg of purified monoclonal antibody was routinelyobtained from each ascites-bearing mouse.

[0182] Further Characterization of Catalytic Activity on Aβ Substrates.

[0183] To fully define the hydrolytic properties of the isolatedanti-transition state antibodies some very important controls can berun. First the ability to completely block catalytic antibody activitywith the appropriate transition state peptide can be verified. Thisnon-cleavable “inhibitor” should bind much more tightly to the antibodycombining sites and thereby prevent substrate binding or cleavage.Substrate specificity can be further established by showing no cleavageof a sham Aβ peptide having a different amino acid sequence. Theproducts of hydrolysis can also be fully characterized by HPLC, aminoacid and mass spectral analysis. Control antibodies that are notdirected against the transition state Aβ can be tested and confirmed toproduce no catalysis. Finally, catalytic activity can be shown to residein the purified Fab fragments of the anti-transition state antibody.

[0184] Purified Anti-Aβ Antibodies Dissolve Preformed Aβ Aggregates.

[0185] (Walker et al., Soc. Neurosci. Abstr. 21: 257 (1995), Zlokovic,B. V., Life Sciences 59: 1483-1497 (1996)). Aβ precipitates were formedand measured in vitro (Yankner et al., Science 250: 279-282 (1990),Kowall et al., Proc. Natl. Acad. Sci. 88: 7247-7251 (1991)). Aradioactive assay was used to quickly screen the different monoclonalantibodies produced for an ability to dissolve preformed Aβ aggregates.After adding ¹²⁵I-Aβ to unlabeled soluble peptide, aggregates wereformed by bringing the solution to pH 5 or by stirring it overnight inPBS. An aliquot of the labeled aggregate was incubated for 1 hr witheither PBS, the 5A11 anti-Aβ antibody or an equal amount of anirrelevant mouse antibody (7D3, anti-human transferrin receptor). Aftercentrifugation, the level of radioactivity in the precipitate wasmeasured.

[0186] Generation of Vectorized Anti-Aβ/Anti-receptor BispecificAntibodies.

[0187] The anti-Aβ antibodies were chemically coupled to anti-humantransferrin receptor and anti-mouse transferrin receptor antibodies bydifferent methods (Raso et al., J. Biol. Chem. 272: 27623-27628 (1997);Raso et al., Monoclonal antibodies as cell targeted carriers ofcovalently and non-covalently attached toxins. In Receptor mediatedtargeting of drugs, vol. 82. G. Gregoriadis, G. Post, J. Senior and A.Trouet, editors. NATO Advanced Studies Inst., New York. 119-138 (1984)).A rapid thioether linkage technique was used to form strictly bispecifichybrids using Traut's reagent and the heterobifunctional SMBP reagent.One component was sparingly substituted with thiol groups (SH). Thesereadily reacted to form a thioether linkage upon mixture with themaleimido-substituted (M) second component following the reaction:

Ab_(A)-SH+Ab_(B)-M→Ab_(A)-S-Ab_(B)

[0188] Gel filtration of the reaction mixture on an S-300 column yieldedthe purified dimer which was 300 kDa and had two sites for binding Aβplus two sites for attachment to transferrin receptors on braincapillary endothelial cells. Non-targeted control hybrids were formed bylinking a nonspecific MOPC antibody to the anti-Aβ antibody. This hybridantibody does bind Aβ, but, being non-reactive with transferrinreceptors, should not cross the blood-brain barrier.

[0189] F(ab′)₂ fragments of the two different antibody types cansimilarly be thioether-linked to form Fc-devoid reagents that cannotbind complement which might otherwise cause neurotoxic effects. Thesesmaller bispecific hybrids (100 kDa) can be formed by reducing theintrinsic disulfides which link the heavy chains of F(ab′)₂ fragments(Raso et al., J. Immunol. 125: 2610-2616 (1980)). The thiols generatedare stabilized and Ellman's reagent (E) is used to activate these groupson one of the components (Brennan et al., Science 229: 81-83 (1985)).Exclusively bispecific F(ab′)₂ hybrids can be formed upon mixing thereduced Fab′ with an activated Fab′ having the alternate specificityaccording to the reaction:

Fab′_(A)-SH+Fab′_(B)-SS-E→Fab′_(A)-SS-Fab′_(B)+E-SH

[0190] Purification on an S-200 column will isolate hybrids with onesite for binding Aβ and one site for interaction with the target epitopeon the brain capillary endothelial cells.

[0191] A similar approach can be used to make even smallerdisulfide-linked single chain Fv heterobispecific dimers,Fv_(A)-SS-FV_(B) (50 kDa), to cross the blood-brain barrier. Soluble Fvscan be constructed to possess a carboxyl-terminal cysteine to facilitatethe disulfide exchange shown in the reaction below, and create 50 kDaheterodimers exclusively:

Fv _(A)-SH+FV_(B)-SS-E→Fv_(A)-SS-FV_(B)+E-SH

[0192] In side by side comparisons between whole antibody and eitherFab′ or Fv based bispecific reagents, the latter have proven to bemoderately more effective on a molar basis for cell uptake via thetransferrin receptor-mediated pathway (Raso et al., J. Biol. Chem. 272:27623-27628 (1997)). Since these smaller constructs are monovalent forthe cell-surface epitope, those findings dispel the notion thatcross-linking of two surface receptors is necessary for the cellularuptake of immunocomplexes.

[0193] Functional Assays for Dual Binding Activity of BispecificAntibodies.

[0194] The capacity of the hybrid reagent to bind ¹²⁵I Aβ was comparedwith that of the parent anti-Aβ antibody in a standard PEG binding assay(see Table 10 for binding assays).

[0195] The ability of the appropriate bispecific antibodies to attach totransferrin receptor bearing human or mouse cells was confirmed bycytofluorimetry. The bispecific antibody was reacted with transferrinreceptor positive human or mouse cells and probed using either a ratIgG-specific or mouse IgG-specific fluorescent secondary antibodyreagent.

[0196] Measurement of Aβ Binding Using ¹²⁵I-Aβ and a Polyethylene GlycolSeparation.

[0197] To ensure bispecificity, hybrid reagents were tested for acapacity to mediate the attachment of ¹²⁵I-Aβ to receptor-bearing cells.Transferrin receptor positive cells were treated with the hybridreagent, washed away unbound material and then exposed these cells to¹²⁵I-Aβ₁₋₄₀. The cells were washed and the amount of cell-boundradioactivity was compared to control cells which had been identicallyprepared except that pretreatment with bispecific antibody was omitted.

[0198] Capillary Depletion.

[0199] The bispecific antibody was labeled with ¹²⁵I and injected i.v.into normal mice. After different lengths of time the mice weresacrificed and the amount of ¹²⁵1-bispecific antibody that crossed theblood-brain barrier and entered the brain was gauged by a mousecapillary depletion method (Friden et al., J. Pharm. Exper. Ther. 278:1491-1498 (1996); Triguero et al., J. Neurochem. 54: 1882-1888 (1990)).The amount of vectorized bispecific antibody found in the brainparenchyma or brain capillary fractions was measured followingdifferential density centrifugation of the brain homogenate. Thesevalues were plotted as a function of time after i.v. injection.Progressive passage from capillaries into the parenchyma indicatesactive transcytosis across the blood-brain barrier.

[0200] Immunoscintigraphy.

[0201] A non-invasive method for monitoring intracerebral deliveryprocess which involves visualizing the entry of a radiolabeledbispecific antibody into the brain of live mice, can also be used.Radiolabeled vectorized bispecific antibody (¹²⁵I-R17/5A11) or anon-vectorized control bispecific antibody were administered to separatemice. Sequential brain images were accumulated at 1, 6, 24 and 48 hoursfollowing i.v. administration of the ¹²⁵I-labeled bispecific antibodyprobes. The animals were chemically immobilized during exposure usingketamine/xylazine anesthesia. This imaging technology could be veryuseful for determining if circulating anti-Aβ antibodies will preventi.v. administered ¹²⁵I-Aβ from entering the brain. Digital scintigraphydata was quantified using standards and the integration functionsprovided in the analysis software.

1 7 1 43 PRT Homo sapiens 1 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr GluVal His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile AlaThr 35 40 2 17 PRT Homo sapiens 2 Asp Ala Glu Phe Arg His Asp Ser GlyTyr Glu Val His His Gln Lys 1 5 10 15 Cys 3 17 PRT Homo sapiens 3 CysTyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val 1 5 10 15Gly 4 10 PRT Homo sapiens 4 Cys Met Val Gly Gly Val Val Ile Ala Thr 1 510 5 14 PRT Homo sapiens 5 Cys His Gln Lys Leu Val Phe Phe Ala Glu AspVal Gly Tyr 1 5 10 6 8 PRT Homo sapiens 6 His Cys Arg His Asn Cys HisArg 1 5 7 6 PRT Homo sapiens 7 His Cys Arg Cys His Arg 1 5

1. An antibody which catalyzes hydrolysis of β-amyloid at apredetermined amide linkage.
 2. The antibody of claim 1 which catalyzeshydrolysis of the amide linkage between residues 39 and 40 of β-amyloid.3. The antibody of claim 1 which catalyzes hydrolysis of the amidelinkage between residues 40 and 41 of β-amyloid.
 4. The antibody ofclaim 1 which catalyzes hydrolysis of the amide linkage between residues41 and 42 of β-amyloid.
 5. The antibody of claim 1 which preferentiallybinds a transition state analog which mimics the transition stateadopted by β-amyloid during hydrolysis at a predetermined amide linkage,and also binds to natural β-amyloid with sufficient affinity to detectusing an ELISA.
 6. The antibody of claim 1 which preferentially binds atransition state analog which mimics the transition state adopted byβ-amyloid during hydrolysis at a predetermined amide linkage, and doesnot bind natural β-amyloid with sufficient affinity to detect using anELISA.
 7. A vectorized antibody which is characterized by the ability tocross the blood brain barrier and the ability to catalyze the hydrolysisof β-amyloid at a predetermined amide linkage.
 8. The vectorizedantibody of claim 7 which is a bispecific antibody.
 9. The vectorizedantibody of claim 8 which has a first specificity for the transferrinreceptor and a second specificity for a transition state adopted byβ-amyloid during hydrolysis.
 10. The vectorized antibody of claim 9which catalyzes hydrolysis of β-amyloid between residues 39 and
 40. 11.A method for sequestering free β-amyloid in the bloodstream of ananimal, comprising the steps: a) providing antibodies specific forβ-amyloid; and b) intravenously administering the antibodies to theanimal in an amount sufficient to increase retention of β-amyloid in thecirculation.
 12. A method for sequestering free β-amyloid in thebloodstream of an animal, comprising the steps: a) providing an antigencomprised of an epitope which is present on endogenous β-amyloid; and b)immunizing the animal with the antigen of step a) under conditionsappropriate for the generation of antibodies which bind endogenousβ-amyloid.
 13. A method for reducing levels of β-amyloid in the brain ofan animal, comprising the steps: a) providing antibodies specific forβ-amyloid endogenous to the animal; and b) intravenously administeringthe antibodies to the animal in an amount sufficient to increaseretention of β-amyloid in the circulation of the animal.
 14. The methodof claim 13 wherein the antibodies specific for β-amyloid are catalyticantibodies which catalyze hydrolysis of β-amyloid at a predeterminedamide linkage.
 15. The method of claim 13 wherein the antibodies aremonoclonal.
 16. The method of claim 13 wherein the antibodies arepolyclonal.
 17. The method of claim 13 wherein the antibodiesspecifically recognize epitopes on the C-terminus of amyloid₁₋₄₃.
 18. Amethod for reducing levels of β-amyloid in the brain of an animal,comprising the steps: a) providing an antigen comprised of an epitopewhich is present on β-amyloid endogenous to the animal; and b)immunizing the animal with the antigen of step a) under conditionsappropriate for the generation of antibodies which bind endogenousβ-amyloid.
 19. The method of claim 18 wherein the antigen is atransition state analog which mimics the transition state adopted byβ-amyloid during hydrolysis at a predetermined amide linkage.
 20. Themethod of claim 18 wherein the antigen is comprised of Aβ₁₀₋₂₅.
 21. Themethod of claim 19 wherein the antibodies generated have a higheraffinity for the transition state analog than for natural β-amyloid. 22.The method of claim 19 wherein the antibodies generated catalyzehydrolysis of endogenous β-amyloid.
 23. A method for preventing theformation of amyloid plaques in the brain of an animal, comprising thesteps: a) providing an antigen comprised of an epitope which is presenton β-amyloid endogenous to the animal; and b) immunizing the animal withthe antigen of step a) under conditions appropriate for the generationof antibodies which bind endogenous β-amyloid.
 24. The method of claim23 wherein the antigen is a transition state analog which mimics thetransition state adopted by β-amyloid during hydrolysis at apredetermined amide linkage.
 25. A method for reducing levels ofcirculating β-amyloid in an animal, comprising the steps: a) providingan antigen comprised of an epitope which is a mimic of a predeterminedhydrolysis transition state of a β-amyloid polypeptide endogenous to theanimal; and b) immunizing the animal with the antigen of step a) underconditions appropriate for the generation of antibodies to the β-amyloidhydrolysis transition state.
 26. A method for reducing levels ofcirculating β-amyloid in an animal, comprising the steps: a) providingantibodies which catalyze the hydrolysis of β-amyloid endogenous to theanimal; and b) intravenously administering the antibodies to the animal.27. A method for preventing the formation of amyloid plaques in thebrain of an animal, comprising the steps: a) providing antibodies whichcatalyze hydrolysis of β-amyloid produced by the animal at apredetermined amide linkage; and b) administering the antibodies to theanimal in an amount sufficient to cause a significant reduction inβ-amyloid levels in the blood of the animal.
 28. A method for reducinglevels of β-amyloid in the brain of an animal, comprising the steps: a)providing vectorized bispecific antibodies competent to transcytoseacross the blood brain barrier, which catalyze hydrolysis of β-amyloidof the animal at a predetermined amide linkage; and b) intravenouslyadministering the antibodies to the animal.
 29. The method of claim 28wherein the vectorized bispecific antibodies specifically bind thetransferrin receptor.
 30. The method of claim 28 wherein the vectorizedbispecific antibodies catalyze hydrolysis of the amide linkage betweenresidues 39 and 40 of β-amyloid.
 31. A method for disaggregating amyloidplaques present in the brain of an animal comprising the steps: a)providing vectorized bispecific antibodies competent to transcytoseacross the blood brain barrier, which catalyze hydrolysis of β-amyloidproduced by the animal at a predetermined amide linkage; and b)intravenously administering the antibodies to the animal in an amountsufficient to cause significant reduction in β-amyloid levels in thebrain of the animal.
 32. A method for disaggregating amyloid plaquespresent in the brain of an animal, comprising the steps: a) providingantibodies which catalyze hydrolysis of β-amyloid produced by the animalat a predetermined amide linkage; and b) administering the antibodies tothe animal.
 33. A method for generating antibodies which catalyzehydrolysis of a protein or polypeptide comprising the steps: a)providing an antigen, the antigen being comprised of an epitope whichhas a statine analog which mimics the conformation of a predeterminedhydrolysis transition state of the polypeptide; b) immunizing an animalwith the antigen under conditions appropriate for the generation ofantibodies to the hydrolysis transition state.
 34. The method of claim33 wherein the protein is β-amyloid.
 35. A method for generatingantibodies which catalyze hydrolysis of a protein or polypeptidecomprising the steps: a) providing an antigen, the antigen beingcomprised of an epitope which has a reduced peptide bond analog whichmimics the conformation of a predetermined hydrolysis transition stateof the polypeptide; b) immunizing an animal with the antigen underconditions appropriate for the generation of antibodies to thehydrolysis transition state.
 36. The method of claim 35 wherein theprotein is β-amyloid.